Efficient haploid cell sorting flow cytometer systems

ABSTRACT

A flow cytometry system ( 1 ) for sorting haploid cells, specifically irradiatable sperm cells, with an intermittingly punctuated radiation emitter ( 56 ). Embodiments include a beam manipulator ( 21 ) and even split radiation beams directed to multiple nozzles ( 5 ). Differentiation of sperm characteristics with increased resolution may efficiently allow differentiated sperm cells to be separated higher speeds and even into subpopulations having higher purity.

I. FIELD OF THE INVENTION

The present invention relates to a flow system for particle analysis.More specifically, the invention relates to the use of a pulsed laser ona flow system for particle analysis which results in more accuratequantification of measurable properties of individual particles. It maybe of particular interest in analyzing populations of very similarparticles, at high speeds, allowing more efficient separation ofparticles into two or more different populations. The invention isparticularly useful in the application of separating live X-chromosomebearing and Y-chromosome bearing sperm of all mammals at higher speeds,better purities and with equal or better sperm health outcomes, meaningless damage to sperm. The invention may contribute significantimprovements to the economics of sperm sorting.

II. BACKGROUND OF THE INVENTION

Lasers can be used to deliver light to biological or non-biologicalparticles and emission spectra can be used for the analysis of particlecharacteristics. In some instances, this can be applied such as where aparticle is self florescent or self color absorbing, is associated byaffinity, avidity, covalent bonds, or otherwise to another moleculewhich may be colored or fluorescent, may be associated to anothermolecule which is colored or fluorescent through a specific biologicalor modeled macromolecular interaction, such as an antibody binding eventor a nucleic acid oligomer or poynucleic acid hybridization event, mayobtain color or fluorescence such as through an enzymatic synthesisevent, an enzymatic attachment or cleavage reaction, enzymaticconversion of a substrate, association of a florescent molecule with anearby quencher, the reaction of a product in certain local proton (pH)or NADH or NADPH or ATP or free hydride (H—) or bound hydride R—(H—)concentrations, or may gain color or fluorescense by way of a variety ofmethods to associate emitted or absorbed light (electromagneticradiation EMR).

Conventional lasers can generate a strong, perhaps intense, source oflight. Through coherence properties of the beam such light may travelvery long distances, perhaps across reflective mirrors which may changethe angle of the light illumination beam, perhaps through prisms orrefractive objects or lenses which may split it into two or more beamsof equal or differing intensity, or may defocus, perhaps expand, orfocus, perhaps concentrate, the beam. Such light may also be affected byfilters which may reduce the net energy of the beam. Most lasers alsoallow the modulation of light intensity, perhaps watts, in the beam byadjustment of an input current from a power supply to the lightgenerating element.

In some applications, conventional lasers used in the analysis andquantification of biological objects can be combined with sensitivelight detectors that may be as simple, such as a photographic film orpaper, or may be more complex, such as a photomultiplier tube. Often, alight detector may collect only information about a cumulative amount oflight, perhaps electromagnetic radiation, EMR, or it may collect andreport on the dynamic changes in intensity of light or EMR hitting allof, or portions of, localized regions of, or positions on the detectorsurface. The light detector may also involve use of a photoelectriccoupling device, which may allow the energy of photons absorbed on theEMR by the light detector to be converted to current proportional to theincident light or EMR on the light detector surface. The photoelectriccoupling device can even be integrated into an electronic circuit withan amplifier which may increase the signal or create gain such that thefluctuations or perhaps summation of amplified current may be availableto an analog or digital logic circuit. Designs may also transmit asignal or data set to a user of a particle analysis instrument and thissignal may be proportional to the static, cumulative, or perhaps evendynamic intensity of the light or EMR incident upon the detector.

In certain uses of laser light to analyze biological particles, adetector may measure the change in intensity of the source light afterincidence upon a particle(s) being analyzed using a reference beam whichtakes a path without incidence upon the particle(s). In other uses oflaser light, modified or unmodified particles take up a fraction of theillumination light or EMR and may emit light of a different frequency.In many cases, the presence of emission light or EMR of a certainwavelength cam be used to identify or to quantify characteristicsassociated with specific particles, or quantitatively measure the amountor number of the specific biological particles present in the sample orin a specific region of or position in the sample.

In some cases, it can be useful to accurately determine very smalldifferences in the illumination light or emission light from two verysimilar biological particles (for example an X-chromosome bearing spermcell versus a Y-chromosome bearing sperm cell). These small differencescan be analyzed by way of serial presentation of perhaps 50,000 separateemission events per second in a liquid stream. These can also bethousands of separate emissions from molecules (nucleic acids orproteins as examples) on an array field allowing analysis of genetic,genomic, proteomic, or glycomic libraries.

The traditional type of laser used for the analysis of particles in flowcytometry is a continuous wave (CW) laser. Often this provides a beam ofconstant intensity. However, in some instances, CW lasers can haveparticular disadvantages for applications as discussed here. The beamcan result in modification or destruction of the sample being observed.For example, with respect to sperm cells, irradiation can result inlower fertility of the sperm cells. Second, in some instances when thelaser beam continuously operates, it may be desirable to have a methodof interrupting the beam if it is moved from a first location ofincidence to a second location of incidence without illumination ofintermediate areas.

In U.S. Pat. No. 5,596,401 to Kusuzawa, a pulsed laser may be used forimaging an object, such as a cell, in a flow cytometer. This disclosuremay be related to improvements in the capture of images from particlessuch as coherence lowering modulations. Kusuzawa may teach a use of acontinuous wave laser for particle detection and imaging.

In U.S. Pat. No. 5,895,922 and U.S. Patent Application No. 2003/0098421to Ho, pulsed laser light may be used to illuminate and detect hazardousbiological particles dispersed in an airflow stream. The invention mayinclude an ultraviolet laser light and looking for the emission offluorescence from potentially hazardous biological particles. Thisdisclosure may teach the disadvantages of a laser diode apparatus.

U.S. Pat. No. 6,177,277 to Soini, describes employing a two-photonexcitation and/or confocal optimal set-up. The invention may relate tothe use of confocal optics to reduce an analysis volume to about 10% ofstandard analysis volume in a flow cytometer. A pulsed laser may provideshort pulses of intense light and may allow the simultaneous absorptionof two photons so that a wavelength of illuminating light beam may belonger than an emitted single photon bursts. Background signal may bereduced by use of a filter. The invention may include dual signalprocessing. The invention as described in Soini, may be beneficial inthe analysis of small particles such as erythrocytes and bacterialcells.

In U.S. Pat. No. 6,671,044 to Ortyn, a special analysis optics andequipment may be used in an imaging flow cytometer. The Ortyn disclosuremay include analyzing a sex of fetal cells in maternal blood as a methodfor determining the sex of a child during early pregnancy. Ortyn mayindicate that analysis rates from an imaging flow cytometer may berestricted to theoretically maximizing at 500 cells per second.

With respect to particle analysis using laser light, the presentinvention discloses technology which addresses each of theabove-mentioned problems.

For the purposes of this invention, a rapidly pulsed, high intensitypulsed laser may be used. This laser may deliver short pulses of highintensity perhaps lasting about 5-20 picoseconds, followed by intervalsbetween pulses which are 100-1000 times as long as the pulses or about0.5-20 nanoseconds. The light may have very high peak intensities overthe period of about 5-20 picoseconds, and low net energies over theperiod of about 2-10 microseconds.

Flow cytometry, using a high-speed cell analysis, or high-speed cellanalysis and sorting instrument, often relies on a laser light source toilluminate a stream of fluid in which particles are entrained. Particlesmay be caused to flow by a point of illumination at a rapid rate, oftenin the range of 500 to 100,000 particles per second. Often the lightfrom the illuminating laser source is of constant intensity. Theparticles in the analysis stream may be of the same size, and may spendthe same amount of time within the area of illumination. The amount oflight illuminating each particle in a large population of particlesanalyzed in series may be identical. A detector may be capable ofmeasuring scattered light, or other types of light emitted by theparticle as a result of auto-fluorescence or fluorescence associatedwith a chemical dye, dye complex, or conjugated dye which may betargeted to one or more types of molecular species contained on orwithin particles in the population and can determine the identity of aparticle and, in some cases, make a measurement of the quantity of aspecific molecular target associated with the particle. A specificmolecular structure on or even within a particle may be characterizedand a quantitative measurement of the amount of associated molecularstructure on or even within a particle, may yield information which maybe used as a basis for sorting out or separating one type of particlefrom another.

In a flow cytometer, there may be a very short time duration between theexact moment that a particle is illuminated and the exact moment that aphysical manipulation or an electrical condition, may be triggered toelicit separation of a specific particle from a stream containingvarious particles. An example of a physical manipulation may be chargingof a droplet. A specific duration may be called a drop delay period, andthe duration may be perhaps as brief as about 100 microseconds orperhaps as long as about 10 milliseconds, and may even be about 1millisecond. In the case of particle sorting, information may bedetected from each particle, computational analysis of the informationmay be determined, and comparison of the computation to a gating valueor perhaps even a selection criteria may be accurately performed withina time period shorter than a duration of the drop delay.

Flow cytometer systems may be useful for measuring an average amount ofa specific molecule present on or even within a population of particles.Past systems may not have measured the exact amount of a specificmolecule on or even within a population of particles. Factors which cancontribute to inaccurate measurements of single particles may includethe saturation of a stain or even a conjugate to a particle, variationin the quanta of illumination light, effects from the shape of aparticle, and perhaps even electronic noise in the detection apparatus.

An example of a particularly challenging problem is the sorting ofX-chromosome bearing and Y-chromosome bearing sperm of mammals at highprocessing rates and high sorting purities. The population of sperm inmost mammals is about 50% X-chromosome bearing and about 50%Y-chromosome bearing. A stain, such as Hoechst 33342, may form complexeswith double stranded DNA. A measurement of total Hoechst 33342-DNAcomplex in each sperm may correlate to the total amount of DNA in eachsperm. In general, mammals have larger X chromosomes than Y chromosomesand may have a differential between total DNA contents of X-chromosomebearing over Y-chromosome bearing sperm for various mammals. Suchdifferentials may include: human having about 2.8%; rabbit having about3.0%; pig having about 3.6%; horse having about 3.7%; cow having about3.8%; dog having about 3.9%; dolphin having about 4.0%; and sheep havingabout 4.2%. The differentials may correlate to a relative difference ofintensities emitted from a stained sperm being sorted for the purpose ofseparation of X-chromosome bearing and Y-chromosome bearing sperm.

Significant achievements have been made in developing stainingconditions to stain DNA in live sperm with Hoechst 33342, such as, theuse of dual orthogonal detection systems to determine sperm orientation,the use of hydrodynamic fluidics to increase the numbers of correctlyoriented sperm, the setting of gain on detectors, and even the use ofhigh-speed electronics. In the most efficient use of said achievements,it may be possible in most mammals to simultaneously sort sperm into twopopulations, X-chromosome bearing, and Y-chromosome bearing, at rates of2500 per second or higher. It may also be possible to sort sperm topurities of 90% or even higher. There may be, however, a distinctproblem in that at rates faster than 2500 per second, the purity of thesample may decrease.

This problem may be understood due to the observation that theco-efficient of variation (CV) in possibly even the best sperm sortingprocedures may be between about 0.7%-1.5%, and with poor conditions caneven be between about 2%-5%. Since the difference in DNA betweenX-chromosome bearing sperm and Y-chromosome bearing sperm in mammals maybe as low as 2.8% as seen in humans and as high as 7.5% as can be seenin chinchillas, the CV may be lower than the DNA differential in orderto achieve a large enough separation of the two populations. Humans haveone of the lowest known DNA differentials and may have some of thelowest known maximum purities in sorting. It may be desirable to improveprocedures which can reduce the CV.

A method which has been shown to improve the CV may be to use higherintensities of laser light illumination. For example, it is known to usecontinuous wave lasers to sort various sperm species with between about100-200 milliwatts of laser illumination, and possibly with about 150milliwatts. It has been observed that doubling or tripling the intensityand increasing the power to about 300-500 mW can improve the CV. Animproved CV can be most apparent by analysis of the “split” between twopeaks on a histogram. Yet, there may be problems associated with anincrease of intensity or perhaps even an increase of power with acontinuous wave laser. In the case of analyzing a Hoechst 33342 DNAcomplex with a continuous wave laser, the light source may be near a UVspectrum and may have some ionizing effect upon the DNA complex.Ionizing may then cause changes to the DNA. Accordingly, sperm sortedwith high intensities continuous wave lasers such as 300-500 mW may notbe as fertile. Another problem may include the energy that it may taketo power a continuous laser to deliver about 150 mW of energy at near UVspectrum. Continuous wave lasers may require 10,000 mW or perhaps evenmore of power. Since there may already be a large amount of electricalpower required to run a continuous wave laser at 150 mW, a much largeramount of power may be required to run a continuous wave laser at higherpowers. Furthermore, a tube life of ion lasers may be reduced whenoperating at higher powers. An additional problem with the use ofcontinuous wave lasers may be that the CV may drop significantly whenusing lower powers such as between about 20-80 mW.

In embodiments, the present invention provides flow cytometer designswhich may incorporate the use of 2 or more flow nozzles, and even asmany as dozens of flow nozzles, possibly operated by a single sortinginstrument. Fields such as microfluidics, optics, electronics, and evenparallel processing may be explored. In other embodiments the presentinvention includes the use of beam splitters to create multiple lightbeams. Yet, a major problem facing the development of reliable flowanalysis and flow sorting in parallel may be the high intensity of laserlight needed for analysis at each nozzle. This problem is particularlyrelevant for applications which require a very low CV in measurement ofidentical particles.

There is a need to provide flow systems for the analysis and sorting ofparticles that require a low CV value, yet may require higher laserlight intensities, yet higher intensities may have negative effects onsperm and require higher power. In the search for solutions to theproblems in flow systems for the analysis and sorting of particles, thefield of pulsed lasers represents a possible solution.

Surprisingly, even though sperm sorted on a high speed flow cytometermay be damaged by UV light between about 300-500 mW, it is now shown inthis invention that powers between about 100-500 mW may not be damagingto sperm if they are delivered in pulses. In embodiments, this mayinclude a pulse having a peak intensity possibly as much as 1000 timeshigher than the intensity of a continuous wave laser. Pulsed lasers maybe designed as quasi-continuous wave lasers and may have fast repetitionrates such as between about 50-200 Megahertz and even up to 80Megahertz. In embodiments, pulses may be between about 5-20 picoseconds.Pulsed lasers may be ideal for providing pulsed light to a stream ofparticles being analyzed in a flow cell or a flow cytometer. Particlesanalyzed in flow cytometers with event rates possibly between10,000-100,000 Hertz, and even between 20,000-60,000 Hertz, may beilluminated from a few hundred pulses from a laser having repetitionrates near 80,000 Hertz. Each pulse may provide an intense amount ofenergy.

There may be certain industrial uses of flow cytometers, as preparativeinstruments, which may be economically limited by the traditionalmethods of processing. It may be desirable to provide systems whichfacilitate parallel processing for industries such as those that rapidlyprocess mammalian ejaculates for the production of large numbers of livesperm for insemination, those that process blood samples for therecovery of specified cells such as fetal cells, white blood cells, stemcells, hematopoetic cells from bone marrow, and the like. In anembodiment of the present invention, special forms of pulsed laser lightcan allow a single laser to illuminate a plurality of nozzles, perhapseven while not reducing the CV of the samples analyzed.

As a result, by the use of special forms of pulsed laser light, furtherimprovements in the speed and sample purity can be seen. These types oflasers may be essential in the design and development of new flowcytometers perhaps having multiple sorting streams as well.

III. SUMMARY OF INVENTION

Accordingly, a broad object of the invention may provide a particleanalysis system having a pulsed laser which can be operated at a lowpower.

Another broad object of the invention can be to provide a particleanalysis system having a pulsed laser which may allow detection ofsmaller differences in illumination or emission to differentiate aparticle characteristic.

Yet another broad object of the invention can be to provide a particleanalysis system having a pulsed laser which allows differentiatedparticles to be separated into subpopulations having a higher incidenceof the desired characteristic.

Another broad object of the invention can be to provide a particleanalysis system having a pulsed laser which allows multiple particledifferentiation systems to be run simultaneously using a single laser.

Another broad object of the invention can be to provide a particleanalysis system having a pulsed laser which affords greater resolvingpower than conventional particle analysis systems using a CW laser.

Another broad object of the invention can be to provide a particleanalysis system having a pulsed laser which generates from fluorochromesupon irradiation greater light intensity than conventional particleanalysis systems using a CW laser.

Another broad object of the invention can be to provide a particleanalysis system having a pulsed laser which allows differentiatedparticles to be separated into subpopulations at a greater rate thanconventional particle analysis systems using a CW laser.

Another broad object of the invention can be to provide a particleanalysis system having a pulsed laser which allows sperm cells of anyspecies of mammal to be differentiated with increased resolution intoX-chromosome or Y-chromosome bearing subpopulations. The benefits ofthis object of the invention may allow differentiation of sperm cellshaving: less DNA bound fluorochrome, less residence time in stainingprotocols, greater elapsed storage time prior to sorting, or perhapseven less affinity to stain due to having been frozen prior to stainingprotocols.

Another broad object of the invention can be to provide a particleanalysis system having a pulsed laser which allows sperm cells to beseparated into X-chromosome or Y-chromosome bearing subpopulationshaving higher purity or separated into X-chromosome or Y-chromosomebearing subpopulations at a greater number per second.

Yet another broad object of the invention can be to provide aminiaturized and parallel flow cytometer which allows a multiple ofnozzles sorting in tandem to be positioned on the same apparatus, thatmay allow increases in the production rate of sorting, by increasing thenumber of nozzles which are sorting on a single apparatus.

Naturally, further independent objects of the invention are disclosedthroughout other areas of the specification.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a block diagram of an embodiment of a particle analysissystem invention which includes beam manipulators such as opticalelements, splitters, filters, directional or the like.

FIG. 2 provides other embodiments of beam manipulators.

FIG. 3 represents a fluidically connected system according to certainembodiments of the invention.

FIG. 4 is a simplified representation of a representative pulse ofradiation that may used in some embodiments.

FIG. 5 provides illustrations of characteristics of a pulsedillumination beam.

FIG. 6 provides illustrations which may differentiate characteristics ofa pulsed radiation beam from a conventional continuous wave radiationbeam.

FIG. 7 shows an embodiment of the invention representing a sortingprocess having multiple nozzles.

FIG. 8 is a drawing of a flow sort embodiment of the invention.

FIG. 9 provides an expanded diagram showing various embodiments of amultiple nozzle assembly.

FIG. 10 depicts embodiments of certain time intervals and light energyquantities which may be derived from particular properties of pulsedlight.

FIG. 11 is a comparison of a pulsed laser radiation beam and acontinuous wave laser radiation beam.

FIG. 12 is a representation of an embodiment for a sensing routine.

FIG. 13 is a representation of a comparison of aggregate data fromvarious trial data.

FIG. 14 provides histograms and bivariant plots of X-chromosome bearingand Y-chromosome bearing subpopulations of sperm cells using a flow sortembodiment of the invention which provides a 20 mW pulsed laser beamincident to the sperm cells analyzed.

FIG. 15 provides histograms and bivariant plots of X-chromosome bearingand Y-chromosome bearing subpopulations of sperm cells using a flow sortembodiment of the invention which provides a 60 mW pulsed laser beamincident to the sperm cells analyzed.

FIG. 16 provides histograms and bivariant plots of X-chromosome bearingand Y-chromosome bearing subpopulations of sperm cells using a flow sortembodiment of the invention which provides a 90 mW pulsed laser beamincident to the sperm cells analyzed.

FIG. 17 provides histograms and bivariant plots of X-chromosome bearingand Y-chromosome bearing subpopulations of sperm cells using a flow sortembodiment of the invention which provides a 130 mW pulsed laser beamincident to the sperm cells analyzed.

FIG. 18 provides histograms and bivariant plots of X-chromosome bearingand Y-chromosome bearing subpopulations of sperm cells using a flow sortembodiment of the invention which provides a 160 mW pulsed laser beamincident to the sperm cells analyzed

FIG. 19 provides histograms of sperm cells analyzed with a flow sortembodiment of the invention compared to a histogram of sperm cells fromthe same sample analyzed with conventional CW flow sort technology.

V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As mentioned earlier, the present invention includes a variety ofaspects, which may be combined in different ways. The followingdescriptions are provided to list elements and describe some of theembodiments of the present invention. These elements are listed withinitial embodiments, however it should be understood that they may becombined in any manner and in any number to create additionalembodiments. The variously described examples and preferred embodimentsshould not be construed to limit the present invention to only theexplicitly described systems, techniques, and applications. Further,this description should further be understood to support and encompassdescriptions and claims of all the various embodiments, systems,techniques, methods, devices, and applications with any number of thedisclosed elements, with each element alone, and also with any and allvarious permutations and combinations of all elements in this or anysubsequent application.

Referring primarily to FIG. 1, the present invention provides, inembodiments, a radiation emitter used with particle analysis systems. Insome embodiments of the invention, a radiation emitter (12) or even anintermittingly punctuated radiation element may convert electricalcurrent into photons of radiation of a specific wavelength and maygenerate radiation or even laser light for analysis of non-biological orbiological particles. Radiation (11) may enter a chamber or region (22)in which it may be modulated or even modified, such as but not limitedto establishing a coherent wave form. Radiation may be modified by abeam manipulator (21) such as optical elements before it may illuminatea particle or even a particle sample(s).

In some embodiments, beam manipulators (21) such as optical elements maybe used and may be located along a light path. Beam manipulators mayinclude mirrors, optically reflective or even refractive mirrors,partially mirrored surfaces, deflectors, beam splitters, refractiveobjects, lenses, filters, prisms, lenses, or the like. Beam manipulatorsmay modify or even modulate a pulsed laser light by focusing,condensing, de-focusing, expanding, splitting, or the like. Radiationmay be split into multiple beams of equal or perhaps even unequalintensity. A radiation beam manipulator may influence a radiation beamsuch as by adapting a beam or changing a beam as a particular situationmay be needed.

With respect to some embodiments of the invention, positional controlelements (24) can provide positional control over a mirror, lens, prism,filter, or other optically relevant components. A positional controlelement may influence the angle of reflection or even refraction of abeam, and may even ultimately direct a final position of a pulsed laserlight beam on a particle or particle sample(s). A positional controlelement may be a mechanical device which may move a mirror along a path,or it may even be a ratcheting or even a stepped device which canprovide large numbers of predefined angles for an optical element. Apositional control element(s) (24) may be used at any point in a pulsedlaser and even a pulsed light assembly or apparatus to modify a beam ofpulsed laser light. An oscillator may provide a constant vibration in anoptical element and may define a frequency and amplitude.

In order to provide splitting of radiation into at least two lightbeams, the present invention may include a beam splitter. This maysubject sperm cells to a reduced power of radiation than a power ofradiation with was originally emitted from a laser source such asradiation emitter. Examples of reduced radiation may include splitting aradiation beam in half, in a fourth of said originally emitted power oreven in an eighth of the originally emitted power, as may be representedby FIG. 2. Of course, there are many options in which to reduce powerand all are intended to be included within the scope of this disclosure.With a pulsed laser, for example producing a 1000 mW output split into 8equal beams of approximately 120 mW, it may be possible to have 8 flowcytometry nozzles sorting together from one light source.

As to other embodiments of the invention, independent beams of pulsedlaser light may be equal or may even be unequal in intensity. Splitbeams can be derived from a source beam of pulsed laser light by theaction of optical splitters. As shown by FIG. 2, a beam splitter (31)such as a prism can divide a beam of radiation into at least two beamsof the same frequency and pulse rate, but may have equal or even unequalintensities. Split beams of light may have intensities which can be eachless intense than, or even additively close to an equivalent to theoriginal beam which entered a splitter or prism. A complex beam splitteror prism, which with multifaceted, three dimensional geometries cansplit a beam of pulsed laser light into more than one, certainly two,possibly dozens or even hundreds of independent beams of pulsed laserlight.

Complex beam splitters (34) may include a three dimensional packed arrayof simple splitters or even prisms which can create combined threedimensional geometries of refraction and reflection and can even split abeam of radiation into more than one, certainly two, possibly dozens oreven hundreds of independent beams of pulsed laser light. This maydiffer from a complex beam splitter in that an array of simple splittersand even prisms may allow individual geometries of each component,refractive index of each component, which may allow a much larger numberof options. A rotational beam splitter (35) can be rotated on an axis,potentially at very high speeds, such that an extremely large number ofpulsed laser light beams may be created to the point where one can nolonger speak of actual beams, but rather of each pulse moving in anindependent direction, slightly different that the prior pulse. Beamsplitters or optical splitters may be used at any point along beam ofpulsed laser light.

Another example of a beam manipulator (21) may include filters can beplaced in a beam path to modulate or modify a property of a pulsed laserlight, and may even reduce the net energy of a particular beam. A largenumber of filters may be used in series or even in parallel across manydifferent beams of pulsed laser light.

A radiation emitter (12) or even a intermittingly punctuated radiationemitter may be embedded in an integrated fashion into a particleanalysis or particle separation system. Alternately, with respect tosome embodiments of the invention, a pulsed laser source may beindependent and splitters, as described above, may be used to split anoriginal beam of light to provide illumination light to numerousindependently operating particle analysis components.

It is understood that a fundamental unit of illumination may be onesingle pulse of a laser light, and that each pulse or laser light may besplit numerous times. Through splitting, filtration or even both, a netenergy of any given pulse illuminating a biological object or sample maybe perhaps as small as a single photon of light or perhaps as large asan original pulse energy emitted from a laser unit.

It is also understood that the use of splitters, which can divide laserlight beams into two or more beams, may increase the number of lightbeams that can illuminate particles, such as biological objects. Inembodiments, a number of pulsed light beams per second that can bedirected toward particles can be multiplied with a splitter. It is alsounderstood that the pulses per second may be altered to a desired numberof pulses per second, timing of the pulse, and even position the pulse.A pulsed light may be distributed by an apparatus to possibly millionsof particles or particle sample(s) which may be located in differentpositions. Through the use of harmonically synchronized oscillations,rotations, and even geometries, many pulses per second may be deliveredto the same particle, sample, or even biological objects. It isparticularly understood that, in embodiments, systems may be establishedwith recurring illumination events.

An embodiment of the present invention may include a system fluidiclyconnected system, such as a flow cytometer system, as seen in FIG. 3.This may be representative of a multiple number of flow cytometrysystems that are linked as one system.

Radiation emitters and even intermittingly punctuated radiationemitters, as described in more detail below, may provide one, two, threeor perhaps even more radiation beams having specific frequencies,wavelengths, intensities, and even watts to illuminate the type ofparticles to be analyzed. An intermittingly punctuated radiation emittermay multiply subject radiation to, for example, sperm cells for a firstamount of time and may multiply terminate radiation of sperm cells for asecond amount of time (41). This may be represented by FIG. 4.

In embodiments, a first amount of time (40) may include an amount oftime radiation occurs and this time may be between about 5 to about 20picoseconds. A second amount of time (41) may be a radiation off timeand may be between about 0.5 to about 20 nanoseconds. Of course, otheramounts of time for each of a first amount and a second amount of timemay be used and all are understood to be included within the scope ofthis invention. A cycle of a first and second time may be understood asa repetition (42). Each repetition may include a time of about 2 toabout 10 microseconds, yet the repetition may vary. In embodiments, arepetition rate may include between about 50 to about 200 MHz and mayeven include a rate up to about 80 MHz. Other repetition rates arepossible and all are meant to be included within the scope of thisdisclosure. In embodiments, a radiation emitter may be a Nd:YAG,Nd:YVO₄, or the like radiation emitters.

FIG. 4 shows parameters taken into consideration when discussing thedifferences between continuous wave (CW) lasers or even gas and pulsedlasers or even solid state lasers. As shown by FIG. 4, an intermittinglypunctuated radiation emitter (56) may emit radiation that may not becontinuous, yet be in short regular pulses which may have a durationtime or a first amount of time (40). Following a pulse, there may be adark period or a second amount of time (41) in which no light may begenerated. The total elapsed time between two pulses, a repetition rate,may therefore be the duration time in addition to the dark period. Apulse line width and dark period may be similar in length, and it couldbe postulated that a laser may actually be illuminating a sample forsomewhat less than “half of the time”. Alternatively, a pulse line widthmay be much smaller than a dark period, and thus a sample may beilluminated for only a very small fraction of the time.

Peak energy (36) and a full amount of energy or joules delivered fromone pulse of laser light is represented in FIG. 5. Fractional amounts(37) of that energy can be split as described above or put through aneutral density filter. Importantly, one may diminish the amount oflight in one pulse used to illuminate a particle by dividing orfiltering the beam. For example, a 350 mW beam can be split into 10equal beams of approximately 35 mW to run 10 independent analysismachines from a single source laser. In practice, the quality ofanalysis at 35 mW must afford information regarding the characteristicsof the particle illuminated, and for commercial applications perhapsafford at least the same amount of information as when particlesilluminated with a CW UV laser running at the standard of 150 mW. FIG. 5may further help in an understanding of the block diagram represented inFIG. 4. Each radiation pulse may be reduced in energy as previouslydiscussed.

An energy density or even watts may be needed to achieve maximum lightemission (38) from a particle upon illumination as shown by FIG. 6. Ithas been contemplated that light input of a continuous wave laser,however, may be so low that a particle may never be fully saturated withillumination (43). An emission light (44) from particles illuminated bya CW laser at a given energy intensity may be constant, as the sourcelight may be constantly refreshing the particle to a certain partialsaturation value. By comparison, emission light (39) from the sameparticle which has been illuminated with pulses may be greater than froma CW laser. Pulses may be short and may have illumination lightintensity many orders of magnitude higher than the illumination level ofa CW laser. It has also been speculated that a fate of light emissionfrom a particle or particle sample(s) during the dark period may bedependent upon the half-life of emission for the illuminated particleand may even be dependent upon the length of time of the dark period. Inthe case where a half life may be as long or longer than the darkperiod, the emission could remain close to maximum during the entiretime across all pulses delivered to the sample.

It should also be pointed out that if instead of reducing the inputenergy by splitting or filtering, one instead uses movement of mirrorsand reflectors, one may reduce the number of pulses delivered to abiological sample to as low as one pulse, while retaining the verystrong intensity of the pulse. Thus, it is a unique aspect of theinvention to provide movement of the full strength pulsed laser beamacross a plurality of particles which may for example be entrained in afluid stream which passes through a flow cytometry nozzle or located onan array or matrix (such as a DNA or protein microarray), or acombination of both, as would be the case of a single laser beingoscillated so that it illuminates a small number of flow cytometrynozzles in close proximity.

Now referring primarily to FIG. 8, irradiatable sperm cells may beintroduced through a sperm sample injection element (4) which may act tosupply irradiatable sperm cells for flow cytometry analysis.Irradiatable sperm cells may be deposited within a nozzle (5) in amanner such that the particles or cells are introduced into a sheathfluid (3). A nozzle may be located in part below an injection point ofsperm cells. A sheath fluid (3) may be supplied by a sheath fluid source(46) through a sheath fluid port (2) so that irradiatable sperm cellsand sheath fluid may be concurrently fed through a nozzle (5).Accordingly, the present invention may provide establishing a sheathfluid and flowing a sheath fluid into a nozzle, and injectingirradiatable sperm cells into a sheath fluid as shown in FIG. 8.

Further, in embodiments, the present invention may include subjectingirradiatable sperm cells radiation. Radiation may be produced from aradiation emitter (12) as discussed previously. In embodiments theradiation emitter may be a intermittingly punctuated radiation emitteror may even be a continuous wave laser.

In embodiments, the present invention may include multiply subjectingsperm cells to radiation having a wavelength appropriate to activatefluorescence in a sperm cell. The invention may include a fluorescenceactivation wavelength. Such wavelength may include about 355 nm. Ofcourse, this may include any wavelength that may be needed to activatefluorescence. Such other wavelengths may include 350 mm, 360 nm andother wavelengths and all are meant to be included within the scope ofthis disclosure.

In embodiments, the present invention may include sufficiently hitting asperm with radiation to cause an irradiatable sperm to emitfluorescence. This may include providing radiation at certainwavelength, power, energy and the like that is enough to cause anirradiatable sperm to emit fluorescence.

In embodiments, the present invention may provide for excitingirradiatable sperm cells that have been subjected to radiation. When inan excited state, the cells may emit fluorescence. In embodiments,irradiatable sperm cells may be multiply excited with radiation. Thismay include radiation that is emitted from a intermittingly punctuatedradiation emitter.

A pulse of laser light may illuminate particles or even a particlesample(s) at a specific location with an EMR frequency or Hertz, timingsuch as a clock, intensity or even watts, and even net energy or joules.The particles may absorb the pulsed light, may get excited and may evenemit light of the same frequency as that of the pulsed laser light, suchas a scatter or may even emit a light of a difference frequency or evenfluorescence. The exact nature of the amount of energy absorbed by aparticle may be related to the chemical properties of the particle, thechemical properties of any objects attached to or closely associatedwith the particles, the physical chemical properties of the particleenvironment, such as biological segregations including membranes,organelles, solutes, pH, temperature, osmolality, colloidal character,or the like, and may even be related to the frequency and intensity ofthe laser light illuminating the particle. An EMR light emission from aparticle, characterized by a wavelength and quantity, can provide highlyaccurate information about the status of a particle when a pulse ofilluminating light is incident. Depending on the nature of the particleand the particle environment, the particle may then emit a florescentlight signal, and may do so over a certain period of time defined by ahalf-life of emission. Typically, a number of pulses of laser light canilluminate a particle or particle sample in a specified period of time,and there can be a corresponding dynamically changing emission of lightover the same period, or a time period after illumination.

Emitted fluorescence from each of the sperm cells may be detected with adetection system (23). A detection system may include a fluorescencedetector (7) which may be connected to a processing unit (15). Whileprocessing the emitted fluorescence, the present invention may includeevaluating the emitted signals. Evaluation of emitted fluorescence mayinclude how much fluorescence may be emitted possibly by comparisonbetween the cells or may even possibly be compared to a predeterminednumber. The present invention may include, in embodiments, selecting anelectrical condition to be associated with each of the sperm cells in asheath fluid flow. An electrical condition may be a charge, voltage orany electrical condition. A drop charge circuit (8) may charge a streamof cells and sheath fluid based upon deduced properties of each of theexcited cells. For example, this may be to charge all of theX-chromosome bearing sperm cells with a positive charge, and chargingall of the Y-chromosome bearing sperm cells with a negative charge. Ofcourse, while the disclosure focuses primarily upon sperm cells, otherparticles may be analyzed as discussed in the various embodimentsdisclosed.

A charged drop may be formed and isolated in a free fall area. A dropmay be based upon whether a desired cell does or does not exist withinthat drop. In this manner the detection system may act to permit a firstand second deflection plates (18) to deflect drops based on whether ornot they contain the appropriate cell or other item. The deflectionplates may be disposed on opposite sides of a free fall area in which adrop may form and the deflection plates may be oppositely charged. As aresult, a flow cytometer may act to sort cells by causing them to landin one or more collectors. Accordingly, by sensing some property of thecells or other items, a flow cytometer can discriminate between cellsbased on a particular characteristic and place them in the appropriatecollector. In some embodiments, X-bearing sperm droplets are chargedpositively and deflected in one direction, and Y-bearing sperm dropletsare charged negatively and deflected the other way. A wasted streamwhich may be unsortable cells may be uncharged and may be collected incollector, an undeflected stream into a suction tube or the like.

In this manner, a sheath fluid may form a sheath fluid environment forthe sperm cells to be analyzed. Since the various fluids are provided tothe flow cytometer at some pressure, they may flow out of nozzle (5) andexit through a nozzle orifice (47). By providing some type of oscillator(6) which may be very precisely controlled through an oscillator control(45), pressure waves may be established within the nozzle andtransmitted to the fluids exiting the nozzle at nozzle orifice. Since anoscillator may act upon the sheath fluid, a stream (19) exiting thenozzle orifice (47) can eventually and regularly forms drops (9).Because the particles or cells are surrounded by the fluid stream orsheath fluid environment, the drops (9) may entrain within themindividually isolated particles or cells, such as sperm cells withrespect to some embodiments of the invention.

In other embodiments, since the drops can entrain particles or cells,the flow cytometer can be used to separate particles, cells, sperm cellsor the like based upon particle or cell characteristics. This isaccomplished through a particle or cell detection system (23). Theparticle or cell detection system involves at least some type ofdetector (7) which responds to the particles or cells contained withinfluid stream. The particle or cell sensing system may cause an actiondepending upon the relative presence or relative absence of acharacteristic, such as fluorochrome bound to the particle or cell orthe DNA within the cell that may be excited by an irradiation sourcesuch as a radiation emitter (12) generating an irradiation beam to whichthe particle can be responsive. While each type of particle, cell, orthe nuclear DNA of sperm cells may be stained with at least one type offluorochrome different amounts of fluorochrome bind to each individualparticle or cell based on the number of binding sites available to theparticular type of fluorochrome used. With respect to spermatozoa, theavailability of binding sites for Hoechst 33342 stain is dependant uponthe amount of DNA contained within each spermatozoa. BecauseX-chromosome bearing spermatozoa contain more DNA than Y-chromosomebearing spermatozoa, the X-chromosome bearing spermatozoa can bind agreater amount of fluorochrome than Y-chromosome bearing spermatozoa.Thus, by measuring the fluorescence emitted by the bound fluorochromeupon excitation, it is possible to differentiate between X-bearingspermatozoa and Y-bearing spermatozoa.

As a result, the flow cytometer acts to separate the particle or cellsby causing them to be directed to one or more collection containers. Forexample, when the analyzer differentiates sperm cells based upon a spermcell characteristic, the droplets entraining X-chromosome bearingspermatozoa can be charged positively and thus deflect in one direction,while the droplets entraining Y-chromosome bearing spermatozoa can becharged negatively and thus deflect the other way, and the wasted stream(that is droplets that do not entrain a particle or cell or entrainundesired or unsortable cells) can be left uncharged and thus iscollected in an undeflected stream into a suction tube or the like asdiscussed in U.S. Pat. No. 6,149,867 to Seidel, hereby incorporated byreference herein. Naturally, numerous deflection trajectories can beestablished and collected simultaneously.

Irradiatable sample cells may include a sample cell that is capable ofemitting rays of light upon illumination. This may or may not includehaving stain molecules attached to a sample particle. Some particles mayhave features that allow them to emit fluorescence naturally withouthaving to add a stain to them.

Laser light incident upon the particle(s) being analyzed may generate atleast one, or perhaps even two, three or more beams of scattered lightor emitted light having specific frequencies, wavelengths, intensitiesand perhaps even watts. All or a portion of the scattered light or evenemitted light may be captured by a detector. A detector may include aphotomultiplier tube or the like detectors.

A detection system may be used to detecting an amount of emittedfluorescence from each of the sperm cells in a flow cytometry system. Adetection system or even a sperm cell fluorescence detector may includea photomultipler tube. In other embodiments a detection system mayinclude an optical lens assembly, a photomultipler tube and even somesort of analysis system such as a computer. An optical lens assembly maycollect emitted fluorescence and transport a collected signal to aphotomultipler tube. The signal detected by a photomultipler tube maythen be analyzed by a computer or the like devices.

A single or perhaps even a multiple digital or analog detector(s) mayreceive all or even a portion of the scattered light or emission light.Analog or digital processor(s) may convert the signals detected by thedetector(s) into analog current or even digital information. Theinformation may accurately represent the identity, frequency, quantity,and even joules of light or EMR received by the detector.

In embodiments, a detector may generate a current or digital signalcorresponding to the amount of light quanta hitting the detector.Certain embodiments of the invention, can provide a one dimensionaldetector which summates all light of the specified wavelength incidentupon the detector surface during a specified period of time. Theduration or even a total time of detection may be as simple as a fullyadditive collection or even integration over an entire analysis time ofthe sample, or it may be a dynamic data set which may record an emissionof light from a biological object(s) or sample(s) over a time period.That time period may be as short as the time between two pulses of thepulsed laser light, or it may be as long as many millions of pulses. Itis understood that such a detector may become two dimensional when thesecond dimension of time is considered.

In other embodiments, a two or three dimensional detector may comprise aflat panel, a three dimensional matrix of unit cells or even pixelswhich can detect an emission light once or more times and can record orreport the interaction specific to that individual unit cell. Theinformation relevant to the operator of an apparatus may be a summationor display of the results of many unit cells. This type of detector mayinclude, but is not limited to, photographic film, photographic paper,or even a microchip capable of sending data for imaging on a televisionscreen or computer screen. It is understood that a two dimensionaldetector may become three dimensional, and three dimensional detectormay become dimensional when the additional dimension of time may beconsidered.

A signal generated by a detector can be processed to provide simpleoutcomes such as photos. A signal generated by a detector may even beable to allow analysis of many thousands or even millions of biologicalobjects in real time with computer graphics which can giverepresentations to allow a user to modulate or modify an analysisprocess in real time. In other embodiments, it may be desirable to sortwith a magnetic detection.

In embodiments, the present invention may include quantitativelydetecting an amount of emitted fluorescence from each of the spermcells. The quantity of the emitted fluorescence may be detected with asperm cell fluorescence quantitative detector. Of course this mayinclude other samples. In sperm cells, a X chromosome bearing sperm anda Y chromosome bearing sperm may be distinguished because an Xchromosome bearing sperm may emit a different amount of fluorescencethan a Y chromosome bearing sperm. In other embodiments, if using othersamples besides sperm cells, the present invention may providedistinguishing between a first population of particles and a secondpopulation of particles due to a difference in an amount of fluorescenceemitted from each population of particles. A distinguishing analysis maybe calculated with a detector.

Operating system controlled computer(s) or even graphic user interfacecontrolled computers may use data from an analog or even digitalprocessor(s). A computer may facilitate direct feedback control of alaser and even analytical equipment. A computer may even provide data tosupport a workstation which may give an operator(s) of an analytical orseparation equipment images that may relate to a behavior of the system.This may allow control of the behavior of the system for optimalanalysis, quantification, and even separation of a biological object(s)or even a sample(s).

Auxiliary computational, command equipment or even control equipment mayprovide local network control of analysis, quantification and separationapparatus. Control equipment may communicate in local area networks(LAN) or even wide area networks (WAN) to provide local or perhaps evendistant operators capability to initiate, monitor, control,troubleshoot, download data or even instructions, upload data orinstructions, terminate, and the like. Control equipment may allowoperation of one, two, three or even more apparatus.

Computational subcomponents may correspond to a command and even acontrol which may be integrated into a pulsed laser design andconstruction. In some embodiments, computational subcomponents may beindependent or even integrated parts of an apparatus and may resideoutside a housing of a pulsed laser.

In embodiments, the present invention may provide staining a sperm cellwith a fluorescent dye. A stained sperm cell (or in other embodiments, astained sample) may be stained with fluorochrome and in yet otherembodiments, may be stained with Hoechst bisbenzimide H33342fluorochrome.

In some instances, a large amount of dye may be used to stain a spermcell. Sperm cells contain deoxyribonucleic acid and deoxyribonucleicacid may have many binding sites that stain (dye) molecules may bindwith. Due to the nature of sperm cells, a stained sperm cell may havemany molecules of a dye attached to each binding site of a sperm.

In embodiments, the present invention may include staining a spermsample for a reduced staining time. The staining time may vary due tothe type of stain used and even due to the type of sample used, heresperm. Typically, staining sperm with Hoechst 33324 may take about 40minutes. under other constant conditions. Some examples of a reducedstaining time may include the following:

-   -   less than about 40 minutes.    -   less than about 35 minutes;    -   less than about 30 minutes;    -   less than about 25 minutes;    -   less than about 20 minutes;    -   less than about 15 minutes;    -   less than about 10 minutes; and    -   less than about 5 minutes.        Of course, other stain times are certainly possible and all        should be understood as represented within the scope of this        invention.

In embodiments, the present invention provides distinguishing between aX chromosome bearing sperm and a Y chromosome bearing sperm in a flowcytometer system. A X chromosome bearing sperm may emit a differentfluorescence from said Y chromosome. For example, a X chromosome bearingsperm may contain more DNA than a Y chromosome bearing sperm, thus a Xchromosome bearing sperm may bind to more dye. When illuminated, a Xchromosome bearing sperm may emit a greater fluorescence than a Ychromosome bearing sperm. The difference may provide the ability todistinguish the two chromosome bearing sperms.

The present invention may include, in embodiments, minimally stainingsperm cells with a fluorescent dye. A minimum sperm stain may includeallowing less stain to bind to each sperm. For example, it may take acertain amount of stain to complete attach stain molecules to eachbinding site of a sperm cell. It may be an efficient option if lessamount of stain could be used while maintaining the ability to achieve adesired result, such as the ability to distinguish between two differentcells after radiation. In embodiments, the present invention may includeproviding a percentage of stain. While a percentage of stain may be aslow as 10%, other examples may include about 90%, about 80%, about 70%and about 60%. All stain percentages are understood as included withinthe scope of this disclosure.

A benefit with respect to sorting sperm cells using a pulsed laser canbe a reduction in the amount of stain taken up by sperm cells duringstaining. Because stains or dyes, such as Hoechst 33342, bind with DNAwithin sperm cells, stain has been considered a factor detrimental tothe viability or fertility of sperm cells. Using a pulsed laser flowsort invention, the amount of stain taken up by sperm cells duringstaining can be reduced by 20% over the amounts used with conventionalCW laser cell sorting technology with similar or better resolution ofX-chromosome bearing and Y-chromosome bearing subpopulations. In certainsperm cell samples the amount of stain taken up by the sperm cells canbe reduced to as little as 60% of the amount used with the same cellssorted by conventional CW cell sorting technology.

Any kind of sample or particle may be used in a flow cytometry system. Asample may include usable cells, reproductive cells, haploid cells,sperm cells, delicate sample, non-biological particles, biologicalparticles, or any kind of cell that can be used with a flow cytometersystem. A useable cell may be a cell that can be used for furtherprocessing or analysis after completion through a cytometry system.Specifically, in embodiments, this may include providing a viable cell.Reproductive cells may include cells that can be used to reproduce anorganism or even a mammal and the like. Haploid cells may include thosecells that have a single set of chromosomes, such as sperm cells. Adelicate sample may include a sample that is fragile or may even beeasily damaged such as reduction in viability. A delicate sample mayhave increased sensitivity to certain environments such as the type ofstain, the sorting process and other environments.

Particles can be non-biological particles such as plastic beads,biological particles such as diploid cells, haploid sperm cells, or thelike. It is to be understood that particles are not limited to cells orbeads but can also include other non-biological particles, biologicalparticles, and the like. Particles may include, without limitation: theindividual binding sites or attachment sites of a molecule on thesurface of a cell or other molecule; large molecules (possibly whetheron the surface of a cell or within a cell) such as proteins, singlestranded DNA, double stranded DNA, mRNA, tRNA, DNA-RNA duplexes,combined protein nucleic acid structures such as a ribosomes,telomerases or the like, DNA or RNA polymerases, samino acid synthetase;the active site of an enzyme such as luciferase, peroxidase,dehydrogenase or even cytochrome oxidase which may require cofactorssuch as ATP, NADH or NADPH; free or bound hydride (H—); or even anystructure biological or non-biological that can be entrained in a fluidstream and made incident to an illumination beam to generate scatteredlight or even emission light.

In another embodiment that may contribute to efficiency in a flowcytometry system, the present invention may include subjecting spermcells to low power radiation. While the range of power that may be usedwith a flow cytometry system may vary, some possibilities for low powermay include:

-   -   less than 300 milliwatt;    -   less than 350 milliwatt;    -   less than 200 milliwatt;    -   less than 175 milliwatt;    -   less than 100 milliwatt;    -   less than 88 milliwatt;    -   less than 50 milliwatt; and    -   less than 25 milliwatt.        Again, other powers of radiation are certainly possible and all        should be understood as represented within the scope of this        invention.

In some examples, a Vanguard Laser may be used. The Vanguard Laser ismanufactured by Spectra-Physics and can emit 80 million pulses persecond (80 MHz). LaserForefront, Spectra-Physics, No. 30 (2001). Eachpulse may have line width of about 12 picoseconds, and a repetition rateof about 10 nanoseconds. This may mean that to an approximation, duringa single repetition of 10 nanoseconds, the pulsed laser may illuminate atarget for about 12 of 10,000 picoseconds or about 0.12% of the totaltime. In other words, a sample being illuminated by a pulsed laser maybe spending approximately 99.88% of the time in the dark. This may alsomean that since a pulsed laser may be delivering 350 milliwatts (mW) oftotal power, during the short 12 picosecond pulse, an average of 280Watts may be delivered to a particle. This may be 800 times more intensethan a light from a continuous wave (CW) laser running at 350 mW. Sincereliable sperm sorting can be performed at 150 mW on a standard CW UVlaser, which may represent a factor of 653 fold, it could behypothesized that it may be possible to run a pulsed laser at as low as150/650 or 0.23 mW and still have light intense enough to illuminate asperm.

In embodiments, the present invention may include utilizing at least oneshared resource to process sperm cells. This may help in efficiency ofsperm sorting in flow cytometry systems. A shared resource may include acomputer system, a sheath fluid, an integrated multiple nozzle device,and the like. In embodiments, a shared resource may include utilizingone radiation source. Radiation may be split into at least two beams andeach beam may be directed toward an nozzle and the sample being sorted.In embodiments, the present invention may include one radiation emitterand a beam splitter or may even include one intermittingly punctuatedradiation emitter and a beam splitter. A beam splitter may be any kindof beam splitter as previously discussed.

As discussed above, the use of refractive, or semi-reflective splittersprovides multiple beams of pulsed laser light derived from the originalsource light These beams may have diminished intensity from the originalbeam, but may be able to each be used to analyze separate particles orparticle sample(s). Also discussed above, each beam may be dedicated toan independent analytical or analytical/separation device (for example,but not limited to, a sperm sorting flow cytometer or cell sorter). Insome embodiments of the invention, each light beam corresponding to anindependent instrument can be split into two light beams of equalintensity and one light beam made incident upon the particle to beanalyzed, and the other light beam can provide a reference beam. Bycomparing the two beams, the absorption of source light by the particlemay be measured. Another unique and important attribute of using asingle pulsed laser to supply light to dozens or hundreds or thousandsof independent analysis or separating machines may be that the entirecomplex of instruments served by the single light will be using the samelight, and to the extent that all machines are performing identical orhighly similar activities, it is possible to use the data from allmachines as internal references and standards to each other, and byusing computers or both which can give local (LAN) as well as distant(WAN) access to the data, to allow operators or persons at a distance tomonitor the performance of each machine in real time.

While multiple nozzles may be integrated into one device, separate flowcytometers having only one nozzle may be lined up so that radiation maybe directed to or even split between each nozzle.

In embodiments, the present invention may include flowing at least onesheath fluid and sperm cells into at least two nozzles. By multiplyingthe number of nozzles operating on a single flow cytometer, the amount(number of particles) analyzed and sorted per unit time may beincreased. In the case where the operation of the flow cytometer may bein a production setting representing a saleable product, multiple nozzlemay increase the number of units produced in a single shift by a singleoperator, and thus a reduction in the costs of each unit produced.

By operating a number of nozzles on the same device, a controllinginstrumentation used on the flow cytometer and operators of the flowcytometer may use statistical analysis of the performance (operationdata) of a multiple of nozzles and may use this data for feedbackcontrol of single nozzles within the population of the nozzles beingused. By operating a number of nozzles on the same device, a singlelight source providing a multiple of beams (one or more for each nozzle)may reside on the same mounting as the nozzles and thereby reduce thecomplexities of light paths related to nozzles running on individualmountings, which may be independent of the mounting of the primaryillumination source. By operating a number of nozzles on the same deviceilluminated by multiple beams from a single light source providing thecapital, operating, parts, service, and maintenance costs from a singlelaser may be distributed across a multiple of productive sortingnozzles, and, therefore, reduce costs per unit produced which areallocated to the laser operation.

FIG. 7 shows multiple nozzles (5) which can provide charged drops (9).The multiple nozzles and collector (20) may be arranged so that a numberof selected containers may be less than a number of nozzles. Selectedcontainers may include containers having collected one specific type ofcell, such as all the X chromosome bearing sperm cells. In this figure,the sorted X chromosome sperm cells may be represented by the containers(32). Here there are three selected containers of a selected cell thathave been sorted from four nozzles.

In other embodiments, the present invention provides utilizing collectedsorted sperm for insemination of female mammals and may even provide fora mammal produced through use of a sorted sperm cells produced with aflow cytometer system according to any of the embodiments as presentedin this disclosure.

In other embodiments, the present invention may include individuallycontrolling or even compositely controlling at least two nozzles. Eachnozzle may individually adjusted according to a desired function with anindividual nozzle control, or a plurality of nozzles may be adjustedcompositely with a single nozzle control device that may be connected toeach of the nozzles.

Another way to increase efficiency in a flow cytometry system, thepresent invention includes rapidly sorting said sperm cells. This may beachieved with a rapid sperm sorter or even a rapid particle samplesorter. Sperm may be sorted at any rate. Such possibilities for a sortrate may include:

-   -   greater than 500 cells per second.    -   greater than 1000 cells per second;    -   greater than 1500 cells per second;    -   greater than 2000 cells per second; and    -   greater than 3000 cells per second.        Other sort rates are certainly possible and all should be        understood as represented within the scope of this invention.

In embodiments, the present invention may include a particle samplecollector such as a sperm cell collector. A collector may be multiplecontainers, a combined collector having individual container, or anytype of collector. For example and as shown in FIG. 9, a combinedcollector (63) may include a collector for one type of particle (32),such as X chromosome bearing sperm populations, a container for a secondtype of particle (33) such as Y chromosome bearing sperm populations,and may even have a third container (64) to collect those drops whichmay not have been charged.

It may be important in designing illumination beams to consider that thecloser the illumination source (laser) may be to an analysis point, theless effect any form of movement such as vibrations may have on the pathof the beam. It may be desirable to provide an system which reduces thedistance between and location of all nozzles to within a very smalldistance of each other (for example all within 15 cm), and greatlysimplifies and enables the use of multiple beams from a single laserlight source.

FIG. 9 shows an exploded diagram of components of a flow cytometrysystem combined into a parallel construction where a multiple of nozzlesmay be operated on a single apparatus. Although the diagram depicts sixnozzles, it is exemplary, such that it might as easily have only 2 or 3nozzles, or may have as many as 8 or 10 or 12 or even 24 nozzles side byside.

A multiple of incoming laser beams or radiation (11), which in mostembodiments could be equal beams derived by splitting from an originalsource beam located close to the nozzles, shines onto an analysis pointwhich is defined by the intersection of the beam or radiation (11) witha narrow stream of fluid which emits from the nozzle (5). In someembodiments, the analysis point may be at the focal point (60) of areflective parabolic dish (61) which may reflect emitted light (62) upto the detection surface (58). Unabsorbed laser light which may not beabsorbed may be absorbed by a heat sink, or it may be measured by anadditional detector to determine an exact real time intensity of thebeam. Each nozzle may be equipped with an oscillator (6) which mayprovide a force causing the stream emitting from the nozzle tip ororifice (47) to break into droplets at defined frequencies such as inthe 10,000-100,000 Hz range. Droplets may be charged, and by action of amagnetic field may be separated. In the case of sorting live mammaliansperm for the presence of X or Y chromosomes, there can be 3 streams ofdroplets: a stream containing primarily X chromosome bearing sperm,which may by example be collected in one container (32) on one side of acollector (20), a stream containing primarily Y chromosome bearingsperm, which may by example be collected in another container (33) onthe other side of a collector (20), and a stream containing sperm whichmay be dead and which may be collected a third container (64) in themiddle of a collector (20). In other embodiments, features such as adetection surface (58), parabolic dishes (61), collectors (20) and insome embodiments nozzles (5) and oscillator(s) (6), may be fabricatedinto single parts or group subassemblies, which may be sandwichedtogether to manifest the actual sorting nozzle architecture.

In other embodiments of the present invention, detection surfaces mayhave diameters (57) similar to the diameter of microtiter plate wells,which can be about 5-8 millimeters, and can have distances (59) betweentwo neighboring flow cytometry nozzle tips which are equal to thedistance between two wells, which may be about 12-18 mm.

Now, referring primarily to FIG. 10, certain time intervals and lightenergy quantities which may be derived from the particular properties oflight provided by a pulsed laser are shown. The standard lasers used inmost flow cytometry and particularly in sperm sorting have been ion tubecontinuous wave (CW) lasers which emit a fairly constant light flux,pulsed lasers may deliver the same rate of net light. For example, aswatts is defined as joules per second, we may consider the period of 1second. It may be exemplary that for many applications in flowcytometry, as many as 10,000-100,000 individual events may be analyzedin 1 second, so that each event requires illumination energies of approx1/10,000-1/100,000 joules.

In contrast, the pulsed laser may emit the same net light in regularpulses. In FIG. 15, which represents an arbitrary time axis, each pulseof laser light (68) can emit a certain energy, and have a certainillumination pulse duration. When a pulse may illuminate a particle, afluorescent emission pulse may occur from that particle which can berepresented by an emission curve (65). An emission curve can representsa classic exponential decay function where maximum emission is at thestart and the rate of emission (decay) is along a corresponding halflife curve. Based on some definition of final decay, for example to thepoint where emission is 1/1000 of original emission, or about 10 halflives, an emission pulse duration (67) can be established. There mayalso be a period commencing from the final decay point occurring at theend of the time summated by illumination pulse duration and emissionpulse duration (67) and the beginning of the next illumination pulse,which can be defined as the resting period. The total sum of theseperiods may the period between pulses which may be the interpulse periodand is typically the inverse of the frequency of the laser.

Using a detection surface, it may be possible to analyze the lightoutput emitted from a particle emission pulse and specifically measuringthe summated total of energy from the emission pulse, which may be anintegration of the area (66) under the decay curve (65). Thismeasurement (70) may be stored as an analog electrical charge in acharge storage device such as an appropriate capacitor, of it may beconverted to a digital value (70) which can be stored in a digitalmemory device. Given that the emission pulse occurs as a dynamicemission event, which through a photodiode/amplifier system may betranslated in realtime to an electrical current (or voltagedifferential), measurements of current or voltage at multiple pointsduring the particle emission may allow the derivative of the decay curveto be determined (71). These can be useful values in statisticalanalysis of multiple identical illumination events of the same particle.

In flow cytometry, which is a broad field in which the instant inventionmay be used, particles which are being illuminated by a laser arecommonly flowing in a fluid stream or a flow cell past a fixed pointupon which a laser beam is focused. In FIG. 11, a rate of flow ofparticles past the point of illumination can be a function of the volumeflux of the stream, and the concentration of the particles. Anillumination period (72) of time in which the particle is beingilluminated may be determined by the flow rate and the size of theparticle in the direction of the flow. When the particle may beilluminated by a pulsed laser generating individual emission pulses (73)which can occur in interpulse periods much less than the illuminationperiod (72) of the particle, then a large number of individual emissionpulses may be derived from the particle (74). In contrast, when aparticle may be illuminated during the same period by a continuous wave(CW) laser, there may be a long emission over the period (75), which cancommonly be detected as a peak profile of current over the period. Ameasurement of the emission from particles illuminated by CW lasers area single long analog events without natural internal cut points and soeither the entire value may be integrated, or the event may be sampledat a discreet multiple of times, or segments of the event areintegrated.

In other embodiments, lasers may be used where the illumination pulseduration may be much smaller than an interpulse period which may itselfbe much shorter than the particle illumination period (72). For example,when the Vanguard Laser is used for the sorting of sperm atapproximately 25,000 events per second, the laser which has 80,000,000illumination pulses per second will deliver approximately 3000 pulsesper event, and about 5-10% or 150-300 pulses occur in the particleillumination period (72). Also, the pulse duration is about 10picoseconds (10-11 sec), the interpulse period is about 10 nanoseconds(10-8 seconds), and the pulse emission period is less than 1 nanosecond.

As may be understood from FIG. 12, an illumination pulse may initiatinga sensing routine. The instant invention may use a laser pulses as aninternal clock to the entire analysis system. Advantages are that eachillumination pulse, which is very brief and very strong, can be used toinitiate each clock cycle. Within a single clock cycle, a computationalsubroutine may run which uses the resting period to calculate specificvalues for each illumination/emission event, and cache the result priorto the initiation of the next clock cycle. An analysis of individualparticles could be manifested over a multiple of clock cycles (forexample 150-300), such that statistical analysis of all emission eventsmapping to a single particle may occur, and averaged values related tothe measurement of the quantity particle and the position of theparticle may be cached. The period between a multiple of events, whichmay be dominated by periods without emissions, may be used to map theidentity and distances and using the momentary gating criteria to effectthe sort. Values of operating parameters and results within each sortmay be cached for viewing at the 1 minute level, possibly operatorstatus, and graphic or summations for entire sort runs may be generated.In FIG. 17, using the example of sperm being sorted using a VanguardLaser, the clock cycle may be about 10-8 seconds. Each clock cycleencompasses three periods. The illumination pulse of 10-11 seconds, theemission period of 10-9 seconds, and the resting period of 9×10-9seconds. Each clock cycle (77) occurs approximately 300 times in eachanalysis event of 3×10-6 seconds. Time between analysis events averages2×10-5 seconds. The time between each analysis and the sort (79) isapproximately 5×10-2 seconds. Operators will usually want to observe nethistorical data over the most recent minute (80) and be able to view theprogress/history of data over the entire sample from start of sort (81).

There may be many hierarchical layers of data occurring dynamically. Atthe same time, with a number of nozzles all sorting the same sample atthe same time, there are simultaneous events occurring in each nozzle ateach hierarchical layer. As it would be labor intensive and inefficientfor the operator to control each nozzle, the statistical analysis andalgorithmic mapping should allow the operator to view status, history,and averages of all nozzles in aggregate and note only nozzles which arenot functioning near the mean of the group. The operator also needs touse commands to change the sorting, which should effect all nozzles atone time.

The data may also be shared between control functions across multiplenozzles and over time to allow the system to make automatic adjustmentssuch as: adjusting optical mirror positions to assure equal laser lightin each beam; tracking the performance of each nozzle to make earlyidentification of nozzle occlusion events; tracking the performance ofeach nozzle to identify differential flow rates and even comparing oneor more semen samples with direct comparisons.

All of these various calculations, in real time, can be calibrated veryprecisely in time, as they may all use the very high frequency laserclock. Thus, in the parallelized flow cytometer, a pulsed laser mayserve as an important integration component for all of the data beinggenerated in a multiple of nozzles.

Referring primarily to FIG. 13, since it may be desirable to stainsamples just prior to sorting, sorting a sample for a period of timebefore staining a second sample which may have been sorted and repeatingthis process several times may create samples which have been stainedand sorted at different times, but may be pooled as a single batch.Aggregated data (82) for each sample may be compared across a multipleof nozzles and multiple of sorts (83), for example, from the sameejaculate of a certain bull. Comparisons of the same bull over multipledays (84) and comparisons between various bulls (85) can give a history.The data from this history may reside within the operating system andmay be used to assist operators in choosing staining concentrations ortimes, or to help identify ejaculates which are sorting worse than theirnormal sorting. Also, if high-throughput resort analysis is available,predicted sex ratios versus. actual sex ratios may be determined, andthe aggregated sex ratios may be compared to identify settings andmethods which different operators may be using, or to identify operatorswho are consistently getting lower results. Also, trends in the sortingperformance may become visible which dictate special maintenance such ascleaning of optics, replacement of nozzle, mirror assemblies, or thelike.

Now referring primarily to FIGS. 14 through 18, embodiments of theinvention are shown using a pulsed laser in the context of flow sortingof sperm cells. Various results from experiments run at different powersof radiation beams are shown. The different experiments included 20 mW,60 mW, 90 mW, 130 mW and 160 mW power and each power was created usingneutral density filters. These embodiments can provide high-purity spermsorting for enrichment of X or Y-chromosome bearing sperm cells whichcan even be up to 98% in purity.

In yet other embodiments, the present invention may provide collectingat least two populations of sample particles, more specifically,collecting a sorted population of X chromosome bearing sperm andcollecting a sorted population of Y chromosome bearing sperm. Acollector may be provided to collect each sorted population.Accordingly, the present invention may include a X chromosome bearingsperm collector and a Y chromosome sperm collector. It may be importantto sort and collect each population at a high purity. A high puritysorted population of X chromosome bearing sperm and said Y chromosomebearing sperm may include a percentage of purity. Of course, anypercentage of purity may exist and some examples may include:

-   -   greater than 85% purity;    -   greater than 90% purity;    -   greater than 95% purity;    -   greater than 96% purity; and    -   greater than 98% purity.        Other percentages of purity are certainly possible and all        should be understood as represented within the scope of this        invention.

Typical pulsed lasers having characteristics similar to that set out byTable 1 or Table 2 can be used with the invention. TABLE 1 Vanguard 150mW Output Power Average Output Power [W] 0.15 UV Beam Size [mm] 1 Energyper Pulse [J] 1.875E−09 Peak Power [W] 234-375 Power per cm{circumflexover ( )}2 [W/cm{circumflex over ( )}2] 2.98E+04

TABLE 2 Vanguard 350 mW Output Power Average Output Power [W] 0.35 UVBeam Size [mm] 1 Energy per Pulse [J] 4.375E−09 Power per Pulse [W]546.875 Power per cm{circumflex over ( )}2 [W/cm{circumflex over ( )}2]6.96E+04

FIGS. 14 through 18 show univariate histograms and a bivariate dot plotsfrom sorting of Hoechst 33342 stained bovine sperm cells. Sperm cellssorted were obtained from the same freshly ejaculated bull sperm dilutedto 200×10⁶ sperm cells per mL and incubated in Hoechst 33342 at 34° C.for 45 min.

With respect to the particular histograms and bivariant plots shown byFIGS. 14 through 19, the event rate (illumination of the sperm cells asthey pass through the pulsed laser beam) was established at 20,000events per second. The sort rate (separation of the sperm cellsdifferentiated by analysis) into subpopulations was varied from 850-3500depending on the power used. The results are also set out by Table 3.TABLE 3 Pulsed Laser Power Setting Purity X % Purity Y % 20 mW pulsed96.5 91.0 60 mW pulsed 93.5 85.5 90 mW pulsed 96.0 89.5 130 mW pulsed 96.0 91.0 160 mW pulsed  97.0 93.0

As can be understood from Table 3, using a pulsed laser sperm cells canbe sorted into high purity X-chromosome bearing and Y-chromosome bearingsubpopulations. For each laser power setting between 20 mW and 160 mWsorted subpopulations had a purity of up to 97.0% of the correct sextype.

Now referring primarily to FIG. 19, histograms compare the resolution ofthe same sample of sperm cells using conventional CW flow sortingtechnology and with an embodiment of the flow sorting inventionutilizing a pulsed laser beam. Importantly, pulsed laser illumination ofstained sperm cells provides superior resolution of X-chromosome bearingsperm cells and Y-chromosome bearing sperm cells. This is true even whenthe pulsed laser beam has a power significantly lower than the comparedto conventional CW flow sorter technology. Even when the pulsed power is130 mW, 70 mW or even 20/150 of the power used in the compared to CWflow sorter technology. The histograms of the pulsed laser experimentsshow a cleaner separation of the two peaks as compared to the continuouswave experiments.

In embodiments, the present invention may include providing a highresolution of a sorted population of sperm cells. A higher resolutionmay be indicative of the purity of a sorted population. While manydifferent resolution values may exist; some examples of high resolutionsmay include:

-   -   greater than 7.0;    -   greater than 7.5;    -   greater than 8.0;    -   greater than 8.5;    -   greater than 9.0; and    -   greater than 9.2.        Of course, other resolution values may exist and all are to be        understood as represented within the scope of the invention.

As discussed above, lower laser power analysis of particles, and in thisparticular embodiment of the invention sperm cells, resolves the longstanding problem of having to have a dedicated laser source for eachflow sorter. By reducing the laser power required, even withoutachieving any other benefit, multiple flow cytometers, flow sorters, orcell sorters can be operated using a single laser source. For example,when sorting is accomplished at about 20 mW, a single 350 mW pulsedlaser can be used to provide illumination light for as many as 18separately functioning flow cytometers or flow sorters used to separatesperm cells on the basis of bearing an X or Y chromosome.

Again referring to FIG. 19, another important benefit provided by apulsed laser invention in the context of sorting sperm cells can beincreased resolution of X-chromosome bearing and Y-chromosome bearingsubpopulations even when sperm cell samples, such as those used in thisspecific example, are stored for long periods of time at about 5° C.,such as 18 hours or longer, or frozen and thawed prior to staining andanalysis, the resolution of sorted sperm cells can improved with thepulsed laser invention compared to conventional CW laser technology.

In embodiments, the present invention may include sorting sperm cells ata low coincidence rate. Some examples of low coincidence rates mayinclude:

-   -   less than 4400;    -   less than 4000;    -   less than 3700; and    -   less than 3600.        Again, other low coincidence rates are certainly possible and        all should be understood as represented within the scope of this        invention.

The present invention may include, in embodiments, collecting sortedpopulations of sperm cells at a high collection rate. A high collectionmay increase productivity and even efficiency. Some examples of highcollection rates may include:

-   -   greater than 2400 sperm per second;    -   greater than 2600 sperm per second;    -   greater than 2900 sperm per second;    -   greater than 3000 sperm per second; and    -   greater than 3100 sperm per second.        Other collection rates are possible and all are meant to be        included within the scope of this invention.

In yet other embodiments, the present invention may include detectingsperm cells at an event rate of between about 10,000 to about 60,000sperm cells per second. Of course, an event rate may be greater than10,000 or even lower than 60,000 cells per second.

A benefit with respect to sorting sperm cells using the pulsed laserinvention can be higher sorting speeds. When resolution of a particularsample is increased, the sort rate of subpopulations of sperm cells to agiven purity can be increased.

Another benefit with respect to sorting sperm cells using the pulsedlaser invention can be a higher purity sort. When resolution of aparticular sample is increased, the purity of the subpopulations ofsperm cells can be increased.

A pulsed laser invention may be understood to have application withrespect to any particular particle characteristic which may bedifferentiated by change of illumination intensity or by detectablelight emission upon illumination with a pulsed light beam. While theapplicant has provided specific examples of differentiating the amountof DNA within a cell using the invention, it to be understood that theseexamples are illustrative of how to make and use the invention withregard to the wide variety of non-biological and biological particles,including, but not limited to, viral particles, polyploid cells, diploidcells, haploid cells (such as sperm cells obtained from any species ofmammal such as any type or kind of bovine, ovine, porcine, or equinesperm cells; or sperm cells obtained from any type or kind of elk, deer,oxen, buffalo, goats, camels, rabbits, or lama; or sperm cells obtainedfrom any marine mammal such as whales or dolphins; or sperm cellsobtained from any rare or endangered species of mammal; or sperm cellsobtained from a zoological species of mammal; or sperm cells obtainedfrom a rare or prize individual of a species of mammal; or sperm cellsobtained from an individual of a species of mammal that used to producedairy or meat products. In embodiments, sperm cells may include any typeof sperm cells such as but not limited to, mammals, bovine sperm cells,equine sperm cells, porcine sperm cells, ovine sperm cells, camelidsperm cells, ruminant sperm cells, canine sperm cells and the like.

It is understood that the present invention may exist in uniqueadvantages when combined with other aspects of the various referencesincorporated.

It is also to be understood that these specific examples provided arenot intended to limit the variety of applications to which the inventionmay be used, but rather are intended to be illustrative how to make anduse the numerous embodiments of the invention for application withanalytical devices such as flow cytometers, cell sorters, microarrayanalyzers, imaging microscopes, or microimaging equipment, which mayeasily be built to contain two or more, and perhaps thousands or evenmillions parallel channels for analysis, and in such that each of theseseparate channels is capable to perform the identical or very similarfunction to a single flow cytometry sorting nozzle, they may beconsidered “machines”, and it is understood that even a very smalldevice which could be held in a persons hand, may contain many hundredsor many thousands of such “machines” and only be able to function if theuse of illumination light is similar or identical to the inventionsdescribed herein.

EXAMPLE 1

Purified fixed bull sperm heads (also described as bull sperm nuclei),stained in standard conditions with DNA binding stain Hoechst 33342, areused as a performance standard to calibrate a sperm sorting flowcytometer prior to the sorting of live sperm. A pulsed laser(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nmand 80 MHz illuminates the sample analysis stream in a flow cytometeroperating at standard settings and provides the histogram plot shown inFig. Ex 1. This demonstrates that a standard sperm sorting flowcytometer equipped with the pulsed laser is able to resolve bull spermnuclei into X-chromosome bearing and Y-chromosome bearing populationsusing standard conditions.

EXAMPLE 2

A sample of live bull sperm is stained in standard conditions with DNAbinding stain Hoechst 33342. A pulsed laser (SpectrophysicsVNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHzilluminates the sample analysis stream in a flow cytometer operating atstandard settings sorting said sperm and provides the histogram plotshown in Fig Ex 2. This demonstrates that a standard sperm sorting flowcytometer equipped with the pulsed laser is able to resolve live sperminto X-chromosome bearing and Y-chromosome bearing populations understandard conditions.

The above sample is sorted for collection of X-chromosome bearing sperm,and the sort collection rate is 3800 live X-chromosome bearing spermsecond. A resort analysis of the sample prepared in said manner measuresthe purity of said sorted sample to be 95%. This demonstrates that astandard sperm sorting flow cytometer equipped with the pulsed laser isable to enrich the content of a sperm population from one in whichapproximately 50% of the sperm are X-chromosome bearing sperm, to one inwhich 95% of the sperm are X-chromosome bearing sperm.

Said sorted sperm above are further processed by standard methods forpackaging into artificial insemination straws, are cryopreserved by thestandard freezing method, and are thawed for analysis of motility of thesperm. Percent motility at various points in the procedure is determinedto be: After stain 80%, after sorting 70%, after cooling 65%, afterfreezing and thawing at 0 minutes 45%, 30 minutes after thawing 45%, 120minutes after thawing 35%. This demonstrates that a standard spermsorting flow cytometer equipped with the pulsed laser is able to enrichlive stained sperm samples which appear normal in respect to the spermmotility.

EXAMPLE 3

A sample of live bull sperm is stained in standard conditions with DNAbinding stain Hoechst 33342. A pulsed laser (SpectrophysicsVNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz isequipped with beam splitters and neutral density filters, in fiveseparate conditions, to provide illumination energy beam levels of 160mW (53% of beam power), 130 mW (43% of beam power), 90 mW (30% of beampower), 60 mW (20% of beam power), and 20 mW (6.6% of beam power),respectively, to illuminate the sample analysis stream of a spermsorting flow cytometer operating at standard settings sorting saidstained sperm and providing the 5 histogram plots shown in Fig Ex 3.This demonstrates that a standard sperm sorting flow cytometer equippedwith the pulsed laser is able to clearly resolve live sperm withenergies as low as 60 mW (20% of the beam).

The above samples are sorted at each of the 5 beam energy settings forcollection of X-chromosome bearing sperm and Y-chromosome bearing spermin separate fractions, and the sort collection rate is 850-3500X-chromosome bearing or Y-chromosome bearing sperm second, depending onthe power used, with lower powers associated with lower sort collectionrates. A resort analysis of the samples prepared in said manner measuresthe purity of said sorted samples as shown in Table Ex 3. Thisdemonstrates that a standard sperm sorting flow cytometer equipped withthe pulsed laser delivering beam energies in the range of 20 mW to 160mW is consistently able to enrich the content of a sperm population fromone in which approximately 50% of the sperm are X-chromosome bearingsperm, to one in which 95% or higher of the sperm are X-chromosomebearing sperm, and simultaneously to one in which 90% or higher of thesperm are Y-chromosome bearing sperm. TABLE 4 Beam Energy (Pulsed) %Purity - X % Purity - Y 20 mW 96.5 91.0 60 mW 93.5 85.5 90 mW 96.0 89.5130 mW  96.0 91.0 160 mW  97.0 93.0

EXAMPLE 4

A sample of live bull sperm is stained in standard conditions with DNAbinding stain Hoechst 33342. A pulsed laser (SpectrophysicsVNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz isequipped with beam splitters and neutral density filters, in twoseparate conditions, to provide illumination energy beam levels of 130mW (43% of beam power) and 70 mW (23% of beam power), respectively, tothe sample analysis stream of a flow cytometer operating at standardsettings sorting said stained sperm. As comparison, the same sample isanalyzed on two identical but different flow cytometers operating atstandard settings and equipped with a CW (continuous wave) lasersdelivering 150 mW in both cases. This demonstrates that even with lowerbeam energies a standard sperm sorting flow cytometer equipped with thepulsed laser provides superior resolution capability when compared to asame standard sperm sorting flow cytometer equipped with a standard CWlaser.

EXAMPLE 5

A sample of live bull sperm is stained in standard conditions with DNAbinding stain Hoechst 33342 with the standard concentration of Hoechst33342 being defined as 100% level of stain (control). Two additionalsamples are prepared which are identical except that they are stainedwith 80% or 60% of the amount of Hoechst 33342 stain, respectively. Apulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energyat 355 nm and 80 MHz is equipped with beam splitters and neutral densityfilters, in two separate conditions, to provide illumination energy beamlevels of 150 mW (50% of beam power) and 90 mW (30% of beam power),respectively, to the sample analysis stream of a flow cytometeroperating at standard settings sorting said stained sperm with 3different concentrations of stain used. The resolution betweenX-chromosome bearing and Y-chromosome bearing sperm for these 6conditions are provided in the 6 histogram plots shown in Fig Ex 5. Thisdemonstrates that lesser amounts of Hoechst 33342 stain may be used toprepare sperm samples for sorting on a standard sperm sorting flowcytometer, if higher pulsed beam energies are also used.

EXAMPLE 6

Purified fixed bull sperm heads (also described as bull sperm nuclei),stained in standard conditions with DNA binding stain Hoechst 33342, areused as a performance standard to calibrate a sperm sorting flowcytometer prior to the sorting of live sperm. A pulsed laser(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nmand 80 MHz and equipped with a beam splitter to provide an illuminationenergy beam level of 150 mW (50% of beam power) illuminates the sampleanalysis stream of a flow cytometer operating at standard settings. Saidstained nuclei are analyzed at 20,000 events/second (a rate comparableto the rate used in live bull sperm analysis), as well as at 59,000events/second. The resolution between X-chromosome bearing andY-chromosome bearing bull sperm nuclei for these 2 event rate conditionsare provided in the 2 histogram plots shown in Fig Ex 6. Thisdemonstrates, that for ideal particles such as nuclei standards, theevent rates of analysis may be increased as much as 3-fold with onlymodest loss in the resolution between X-chromosome bearing andY-chromosome bearing bull sperm nuclei.

EXAMPLE 7

Samples of live bull sperm from 4 different bulls were stained instandard conditions with DNA binding stain Hoechst 33342. A pulsed laser(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nmand 80 MHz is equipped with beam splitters and neutral density filters,in two separate conditions, to provide illumination energy beam levelsof 300 mW (100% of beam power) and 150 mW (50% of beam power),respectively, to the sample analysis stream of a flow cytometeroperating at standard settings sorting said stained sperm samples. Ascomparison, the same samples are sorted on an identical but differentflow cytometer operating at standard settings and equipped with a CW(continuous wave) laser delivering 150 mW of energy in the illuminationbeam. The samples are bulk sorted, which means both X-chromosome bearingand Y-chromosome bearing sperm fractions are pooled. The sorted spermsamples are cryopreserved using standard procedures and the percent ofpost thaw sperm motilities, as well as the percent of live and deadusing PI staining with flow cytometry analysis are scored. The averagesfor all 4 bulls with the 3 different illumination conditions are shownin Table 5. This demonstrates without statistical significance that allthree conditions of illumination yield similar numbers of intact viablesperm after sorting. TABLE 5 % Motility at % Motility at % Live % Live 0min 90 min at 0 min at 90 min Laser (mW) post thaw post thaw post thawpost thaw CW (150 mW) 50.0 42.5 43.5 40.6 Pulsed (150 mW) 46.3 42.5 40.037.9 Pulsed (300 mW) 48.1 36.3 40.3 37.2

EXAMPLE 8

Samples of live bull sperm from 5 different bulls are stained instandard conditions with DNA binding stain Hoechst 33342, with thestandard concentration of Hoechst 33342 being defined as 100% level ofstain (control). Two additional samples from the same 5 bulls areprepared which are identical except that they are stained with 80% or60% of the amount of Hoechst 33342 stain, respectively.

A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW ofenergy at 355 nm and 80 MHz is equipped with beam splitters and neutraldensity filters, in two separate conditions, to provide illuminationenergy beam levels of 150 mW (50% of beam power) and 90 mW (30% of beampower), respectively, to the sample analysis stream of a flow cytometeroperating at standard settings sorting said stained sperm samples. Ascomparison, the same samples are sorted on an identical but differentflow cytometer operating at standard settings and equipped with a CW(continuous wave) laser delivering 150 mW of energy in the illuminationbeam.

For the sorting procedures on all these samples, the average values forresolution (higher values are better), the coincidence rates (lowervalues are better), the sort collection rates (higher values are better)are compared and shown in Table 6. This demonstrates that sortingefficiencies in all conditions tested with the pulsed laser were equalto or better than the sorting efficiencies achieved using the standardCW laser. TABLE 6 Co-incidence Stain(%)/Laser (mW) Resolution rate SortRate 100/150 pulsed 8.0 3570 3160 100/90 pulsed 9.3 3560 2610 80/150pulsed 8.2 3600 3160 80/90 pulsed 9.6 3500 2480 60/150 pulsed 8.6 36002940 60/90 pulsed 9.8 3520 2450 100/150 CW 7.6 4380 2720

The same samples are bulk sorted, which means both X-chromosome bearingand Y-chromosome bearing sperm fractions are pooled. The sorted spermsamples are cryopreserved using standard procedures and the percent ofpost thaw motilities, as well as the percent of live/dead using PIstaining with flow cytometry analysis are scored. The averages for all 5bulls with the 7 different stain and illumination conditions are shownin Table 7. This demonstrates that sperm viability and live counts inall conditions tested with the pulsed laser were equal to or better thanthe sperm viability and live counts achieved using the standard CW laserand standard stain. TABLE 7 % Motility at % Motility at % Live % LiveStain(%)/ 0 min 120 min at 30 min at 120 min Laser (mW) post thaw postthaw post thaw post thaw 100/150 pulsed 43.3 34.0 36.8 32.4 100/90pulsed 42.8 33.5 35.3 33.2 80/150 pulsed 42.8 33.8 35.1 35.1 80/90pulsed 42.0 35.3 35.2 29.7 60/150 pulsed 40.8 31.0 35.8 35.1 60/90pulsed 41.0 32.8 34.4 33.7 100/150 CW 39.8 33.3 33.4 28.6

EXAMPLE 9

Samples of live bull sperm from 5 different bulls and 2-6 replicateswere stained in standard conditions with DNA binding stain Hoechst 33342(80%) and bulk sorted under standard conditions in a sperm sorting flowcytometer at event rates of 23,000 sperm/second. A pulsed laser(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nmand 80 MHz is equipped with beam splitter to provide illumination energybeam level of 150 mW (50% of beam power) to the sample analysis streamof a flow cytometer operating at standard settings sorting said stainedsperm samples.

As a control comparison, same samples of live bull sperm from 5different bulls and 2-6 replicates were stained in standard conditionswith DNA binding stain Hoechst 33342 (100%) and these samples weresorted on an identical but different flow cytometer operating atstandard settings and equipped with a CW (continuous wave) laserdelivering 150 mW of energy in the illumination beam.

Said various sperm samples were used at concentrations of 200,000sperm/ml or 1 million sperm/ml to inseminate matured bovine oocytes instandard procedures of in-vitro fertilization (IVF). Cleavage and 2 cellrates at 2.75 days post-insemination, blastocyst development rates at7.75 days post-insemination, total cell numbers in blastocyst and theblastocyst quality at 7.75 days were measured. The average results for587 oocytes inseminated with sorted sperm prepared from the systemequipped with the pulsed laser, and for 558 oocytes inseminated withsorted sperm prepared from the system equipped with the CW laser areshown in Table 8. Note: lower numbers for blastocyst quality are better.This demonstrates that sperm prepared by a standard sperm sorting flowcytometer equipped with the pulsed laser is capable of fertilizingoocytes in standard IVF procedures and exhibits similar cleavage andblastocyst rates, with the mean quality of blastocysts being slightlybetter when inseminated with sperm sorted using the standard flowcytometer equipped with the pulsed laser. TABLE 8 % % % quality of cellcounts in Laser (mW) Cleaved 2 cell blastocyst blastocyst blastocyst CW(150 mW) 50.7 27.3 6.7 2.7 131.8 Pulsed (150 mW) 49.7 29.5 5.2 2.1 136.2

EXAMPLE 10

Samples of live bull sperm from 3 different bulls, on multiple days,were stained in standard conditions with DNA binding stain Hoechst 33342(100%) and bulk sorted under standard conditions in a sperm sorting flowcytometer at event rates of 20-23,000 sperm/second. A pulsed laser(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nmand 80 MHz is equipped with a beam splitter to provide illuminationenergy beam level of 150 mW (50% of beam power) to the sample analysisstream of a flow cytometer operating at standard settings sorting saidstained sperm samples.

As a control comparison, same samples of live bull sperm from the same 3different bulls on same days, were stained in standard conditions withDNA binding stain Hoechst 33342 (100%) and these samples were sorted onan identical but different flow cytometer operating at standard settingsand equipped with a CW (continuous wave) laser delivering 150 mW ofenergy in the illumination beam.

Said various sperm samples were used in amounts of 2 million sperm percyropreserved artificial insemination straw containing 0.25 ml of fluid.

Control straws containing 10 million unsorted sperm, and control strawscontaining 2 million X enriched sperm sorted using a sperm sorting flowcytometer equipped with a the standard CW laser were used.

In a heterospermic analysis, X-fractions from CW laser sorts were mixedin equal sperm numbers with Y-fractions from pulsed laser sorts tocreate the #1 comparision. Y-fractions from CW laser sorts were mixed inequal sperm numbers with X-fractions from pulsed laser sorts to createthe #2 comparison. Identification of sex in fetuses at 60 days was usedas a marker to assign the sex outcome, and accordingly, the likelycondition (which laser) can be attributed to successful fertilization.The heterospermic method is particularly useful, as all other factorsthan sorting procedure are internally controlled in each insemination.

Holstein heifers weighing approximately 750 pounds were synchronizedusing CIDR/Lutalase. Thereafter observed (AM or PM) for standing heatand were inseminated at 12-24 hours after heat observation. Using 2inseminators, and a single deep uterine insemination treatment, with 5test groups spaced approximately one month apart, at a single farm,pregnancy rates and sex of fetus were determined at 60 days postinsemination using ultrasonograhy. The results shown in Table 9demonstrate that the sperm sorted with a standard sperm sorting flowcytometer equipped with a pulsed laser give essentially identicalpregnancy rates as sperm sorted using a standard sperm sorting flowcytometer equipped with the standard CW laser. TABLE 9 % ConceptionPreganancies/ Sperm dose type Rate Inseminations Unsexed controlcontaining 10 million 62.5 55/88 total sperm X-sexed control containing2 million 56.4 101/179 total sperm Heterospermic #1 containing 2 million50.0 45/90 total sperm Heterospermic #2 containing 2 million 58.4 52/89total sperm Pregancies attributed to CW laser 49.50% 48/97 Preganciesattributed to pulsed laser 50.50% 49/97

EXAMPLE 11

A sample of live dolphin sperm was collected at poolside, shipped viaair freight, and stained with Hoechst 33342 approximately 6 hours aftercollection. The sorting efficiencies for the single stained sample werethen tested on two identical Mo Flo SX sperm sorters, in one caseequipped with a pulsed laser (Spectrophysics VNGD350-HMD355) delivering300 mW of energy at 355 nm and 80 MHz equipped with a beam splitter toprovide illumination energy beam level of 150 mW (50% of beam power),and the second case with an Innova 90-6 (CW—continuous wave) laserdelivering 150 mW of beam energy. The dolphin ejaculate was stained 3times, and in each case sorted for approximately 2 hours.

Using the CW laser, with sorter event rates at 30,000/sec an averageco-incidence rate of 6430/sec was observed, X-chromosome bearing spermwere collected at an average rate of 3450/sec and a total of 72 millionsperm were collected in 7 hours for an average recovery of 10.3 millionsperm per hour.

Using the pulsed laser, with sorter event rates at 30,000/sec an averageco-incidence rate of 5400/sec was observed, X-chromosome bearing spermwere collected at an average rate of 3930/sec and a total of 79.5million sperm were collected in 6.33 hours for an average recovery of12.6 million sperm per hour.

The recovered sperm from both samples has X purities of >95% andpost-thaw motility of >50%.

EXAMPLE 12

A sample of live canine sperm was collected from a common dog housed ata kennel and stained about 3 hours later with a non-optimized quantityof Hoechst 33342. The stained sperm were analyzed by a standard spermsorter equipped with a pulsed laser (Spectrophysics VNGD350-HMD355)delivering 300 mW of energy at 355 nm and 80 MHz equipped with a beamsplitter to provide illumination energy beam level of 150 mW (50% ofbeam power). Approximately 43% of the sperm were correctly oriented.From the correctly oriented stained canine sperm, approximately 32% werecollected as X-chromosome bearing sperm, and approximately 36% werecollected as Y-chromosome bearing sperm. Visual inspection by microscopeshowed high numbers of canine sperm in both samples to be motile.

EXAMPLE 13

The standard CW laser uses a cathode tube which requires an averageinput of 12 KW of electrical power, and a large volume of cooling water,or a chiller with a load of approximately 15 KW. The pulsed laser(Spectrophysics VNGD350-HMD355 delivering 300 mW of energy at 355 runand 80 MHz) requires approximately 500 watts (0.5 KW).

The standard CW laser also requires replacement of the cathode tubeafter approximately 5000 hours of use, at a replacement cost of about$12,000, whereas the VNG pulsed laser is expected to have 30,000+hoursof operation before refurbishment of head element at similar costs.

A commercial operation using the sperm sorting flow cytometers to sortsperm for production of artificial insemination straws, running 24 hoursper day, year-round, may be expected to operate lasers for 8,640 hoursper year.

Electric utility and water rates in Fort Collins quoted for the year2004 were used to calculate the operating costs of the standard CWlaser, in the first case cooled by utility water and in the second casecooled using electric powered chiller. The pulsed laser requires nocooling. The comparative costs are shown in Table 10. This demonstratesthat the pulsed laser has significant benefits in reducing the costs ofoperation of a sperm sorting flow cytometer. TABLE 10 CW laser CW laserwith water with electric Pulsed Cost component cooling chiller coolingLaser Electrical Charges $4,389 $9,828 $183 Water Charges $6,483 $0 $0Laser tube or rebuild $20,736 $20,736 $3,456 TOTAL (1 year) $31,608$30,564 $3,639

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. It involvesboth sorting techniques as well as devices to accomplish the appropriatesorting system. In this application, the sorting techniques aredisclosed as part of the results shown to be achieved by the variousdevices described and as steps which are inherent to utilization. Theyare simply the natural result of utilizing the devices as intended anddescribed. In addition, while some devices are disclosed, it should beunderstood that these not only accomplish certain methods but also canbe varied in a number of ways. Importantly, as to all of the foregoing,all of these facets should be understood to be encompassed by thisdisclosure.

The discussion included in this application is intended to serve as abasic description. The reader should be aware that the specificdiscussion may not explicitly describe all embodiments possible; manyalternatives are implicit. It also may not fully explain the genericnature of the invention and may not explicitly show how each feature orelement can actually be representative of a broader function or of agreat variety of alternative or equivalent elements. Again, these areimplicitly included in this disclosure. Where the invention is describedin device-oriented terminology, each element of the device implicitlyperforms a function. Apparatus claims may not only be included for thedevice described, but also method or process claims may be included toaddress the functions the invention and each element performs. Neitherthe description nor the terminology is intended to limit the scope ofthe claims included in this or in any subsequent patent application.

It should also be understood that a variety of changes may be madewithout departing from the essence of the invention. Such changes arealso implicitly included in the description. They still fall within thescope of this invention. A broad disclosure encompassing both theexplicit embodiment(s) shown, the great variety of implicit alternativeembodiments, and the broad methods or processes and the like areencompassed by this disclosure and may be relied upon when drafting theclaims for the full patent application. This patent application seeksexamination of as broad a base of claims as deemed within theapplicant's right and is designed to yield a patent covering numerousaspects of the invention both independently and as an overall system.

Further, each of the various elements of the invention and claims mayalso be achieved in a variety of manners. This disclosure should beunderstood to encompass each such variation, be it a variation of anembodiment of any apparatus embodiment, a method or process embodiment,or even merely a variation of any element of these. Particularly, itshould be understood that as the disclosure relates to elements of theinvention, the words for each element may be expressed by equivalentapparatus terms or method terms—even if only the function or result isthe same. Such equivalent, broader, or even more generic terms should beconsidered to be encompassed in the description of each element oraction. Such terms can be substituted where desired to make explicit theimplicitly broad coverage to which this invention is entitled. As butone example, it should be understood that all actions may be expressedas a means for taking that action or as an element which causes thataction. Similarly, each physical element disclosed should be understoodto encompass a disclosure of the action which that physical elementfacilitates. Regarding this last aspect, as but one example, thedisclosure of a “sorter” should be understood to encompass disclosure ofthe act of “sorting”—whether explicitly discussed or not—and,conversely, were there effectively disclosure of the act of “sorting”,such a disclosure should be understood to encompass disclosure of a“sorter” and even a “means for sorting” Such changes and alternativeterms are to be understood to be explicitly included in the description.

Any acts of law, statutes, regulations, or rules mentioned in thisapplication for patent; or patents, publications, or other referencesmentioned in this application for patent are hereby incorporated byreference. In addition, as to each term used it should be understoodthat unless its utilization in this application is inconsistent withsuch interpretation, common dictionary definitions should be understoodas incorporated for each term and all definitions, alternative terms,and synonyms such as contained in the Random House Webster's UnabridgedDictionary, second edition are hereby incorporated by reference.Finally, all references listed herein and in the table of references aslisted below or other information statement filed with the applicationare hereby appended and hereby incorporated by reference, however, as toeach of the above, to the extent that such information or statementsincorporated by reference might be considered inconsistent with thepatenting of this/these invention(s) such statements are expressly notto be considered as made by the applicant(s). I. U.S. PATENT DOCUMENTSFILING DOCUMENT NO DATE NAME CLASS SUBCLASS DATE 2002/0113965 A1 Aug.22, 2002 Roche et al. 356 339 Oct. 02, 2001 2002/0141902 A1 Oct. 03,2002 Ozasa et al. 422 85.09 Mar. 27, 2002 2002/0186375 A1 Dec. 12, 2002Asbury et al. 356 440 May 01, 2001 2003/0098421 A1 May 29, 2003 Ho 250458.1 Nov. 27, 2001 2003/0207461 A1 Nov. 06, 2003 Bell et al. 436 172Nov. 14, 2001 2003/0209059 A1 Nov. 13, 2003 Kawano et al. 73 53.01 Mar.28, 2003 2004/0005582 A1 Jan. 08, 2004 Shipwash 435 6 Dec. 19, 20023,893,766 Jul. 08, 1975 Hogg 356 36 Jun. 14, 1973 4,362,246 Dec. 07,1982 Adair 209 3.3 Jun. 14, 1980 4,660,971 Apr. 28, 1987 Sage et al. 35639 May 03, 1984 4,988,619 Jan. 29, 1991 Pinkel 435 30 Nov. 30, 19875,088,816 Feb. 18, 1992 Tomioka et. al 356 39 Mar. 06, 1990 5,135,759Aug. 04, 1992 Johnson 424 561 Apr. 26, 1991 5,315,122 May 24, 1994Pinsky, et al. 250 461.2 5,371,585 Dec. 06, 1994 Morgan et al. 356 246Nov. 10, 1992 5,439,362 Aug. 08, 1995 Spaulding 424 185.1 Jul. 25, 19945,466,572 Nov. 14, 1995 Sasaki et al. 435 2 Apr. 25, 1994 5,483,469 Jan.09, 1996 Van den Engh et al. 364 555 Aug. 02, 1993 5,596,401 Jan. 21,1997 Kusuzawa 356 23 Sep. 14, 1994 5,602,039 Feb. 11, 1997 Van den Engh436 164 Oct. 14, 1994 5,602,349 Feb. 11, 1997 Van den Engh 73 864.85Oct. 14, 1994 5,660,997 Aug. 26, 1997 Spaulding 435 7.21 Jun. 07, 19955,690,895 Nov. 25, 1997 Matsumoto et al. 422 73 Dec. 06, 1996 5,700,692Dec. 23, 1997 Sweet 436 50 Sep. 27, 1994 5,726,364 Mar. 10, 1998 Van denEngh 73 864.85 Feb. 10, 1997 5,793,485 Aug. 11, 1998 Gourley 356 318Jan. 13, 1997 5,895,922 Apr. 20, 1999 Ho 250 491.2 May 23, 19975,985,216 Nov. 16, 1999 Rens, et al. 422 73 Jul. 24, 1997 6,149,867 Nov.21, 2000 Siedel, et al. 422 73 Dec. 31, 1997 6,177,277 B1 Jan. 23, 2001Soini 436 63 Jan. 03, 1996 6,263,745 Jul. 24, 2001 Buchanan, et al. Dec.03, 1999 6,357,307 Mar. 19, 2002 Buchanan, et al. 73 865.5 Jul. 20, 20016,411835 B1 Jun. 25, 2002 Modell et al. 600 407 Feb. 02, 1999 6,463,314B1 Oct. 08, 2002 Haruna 600 407 Feb. 19, 1999 6,528,802 Mar. 04, 2003Karsten, et al. 250 459.1 Jun. 01, 2001 6,534,308 B1 Mar. 18, 2003Palsson et al. 435 288.7 Nov. 30, 1999 6,537,829 Mar. 25, 2003 Zarling,et al. 436 514 Dec. 01, 1999 6,577,387 B2 Jun. 10, 2003 Ross, III et al.356 124 Dec. 29, 2000 6,590,911 B1 Jul. 08, 2003 Spinelli et al 372 22Jun. 02, 2000 6,604,435 Mar. 13, 2002 Buchanan, et al. Aug. 12, 20036,618,679 B2 Sep. 09, 2003 Loehrlein et al. 702 20 Jan. 27, 20016,642,018 B1 Nov. 04, 2003 Koller et al. 435 40.5 Mar. 13, 20006,667,830 B1 Dec. 23, 2003 Iketaki et al. 359 368 Apr. 09, 19996,671,044 B2 Dec. 30, 2003 Ortyn et al. 356 326 Nov. 16, 2001 6,673,095B2 Jan. 06, 2004 Nordquist 607 89 Feb. 12, 2001

II. FOREIGN PATENT DOCUMENTS DOCUMENT NO DATE COUNTRY EP 0 288 02920.04.88 Europe WO 96/12171 Apr. 25, 1996 PCT WO 98/34094 Aug. 06, 1998PCT WO 99/05504 Jul. 24, 1998 PCT WO 99/33956 Jul. 08, 1999 PCT WO01/40765 Jun. 07, 2001 PCT WO 01/40765 Jul. 06, 2001 PCT WO 01/85913Nov. 15, 2001 PCT

III. OTHER DOCUMENTS (Including Author, Title, Date, Pertinent Pages,Etc.) Dean, P. N., et al., “Hydrodynamic orientation of spermatozoaheads for flow cytometry”, Biophysical Journal. 23: 7-13, 1978 Elmes, R.S., et al., “Evaluation of the Spectra Physics Vanguard laser as a newUV light source for Flow Cytometry,” Laboratory for Cell Analysis,Comprehensive Cancer Center, University of California, 4pages     (Date) Fulwyler, M. J. 1977. Hydrodynamic orientation ofcells. J Histochem. Cytochem. 25: 781-783. Gurnsey, M. P., and Johnson,L. A., “Recent improvements in efficiency of flow cytometric sorting ofX and Y- chromosome bearing sperm of domestic animals: a review”, 1998,New Zealand Society of Animal Protection, three pages. Johnson, L. A.,et al., “Enhanced flow cytometric sorting of mammalian X and Y sperm:high speed sorting and orienting No. 77.1e for artificial insemination”,Theriogenology. 49(1): 361. abstr., 1998 Johnson L. A., et al., “Flowcytometry of X- and Y- chromosome bearing sperm for DNA using animproved preparation method and staining with Hoechst 333-42”, GarneteResearch 17: 203-212, 1987 Johnson L. A., et al., “Modification of alaser-based flow cytometer for high resolution DNA analysis of mammalianspermatozoa”, Cytometry 7: 266-273, 1986 Johnson, L. A., et al.,“Improved flow sorting resolution of X- and Y- chromosome bearing viablesperm separation using dual staining and dead cell gating”, Cytometry 17(suppl 7): 83, 1994 Johnson, L. A., et al., “Sex Preselection: HighSpeed Flow Cytometric Sorting of X and Y sperm for Maximum efficiency”,Theriogenology 52: 1323-1341, 1999 Johnson L. A., et al., “Sexpreseletion in rabbits: Live births from X and Y sperm separated by DNAand cell sorting”, Bio Reprod 41: 199-203, 1989. Kachel, V., et al.,“Uniform Lateral Orientation, Caused by Flow Forces, of Flat Particlesin Flow-Through Systems”, The Journal of Histochemistry andCytochemistry, 1997, Vol. 25, No. 7, pp 774-780. Lightwave Electronics,“Xcyte”, www.LightwaveElectronics.com, 2 pp. “Introducing the Vanguard—4 Watts of UV from a Quasi-cw, all solid state laser,” LaserForefront,A Monthly Update On The State Of Technologies In The Laser Industry2001, No. 30 Rens, W., et al., “Improved Flow Cytometric Sorting of X-and Y- Chromosome Bearing Sperm: Substantial Increase in Yield of SexedSemen”, Molecular Reproduction and Development, 1999, pp 50-56. Rens,W., et al., “A Novel Nozzle for More Efficient Sperm Orientation toImprove Sorting Efficiency of X and Y Chromosome-Bearing Sperm”,Technical Notes, Cytometry 33, 1998, pp 476-481. Siedel, G. E. Jr.,Herickhoff, L. A., Schenk, J. L., Doyle, S. P. and Green, R. D. 1998.Artificial insemination of heifers with cooled, unfrozen, and sexedsemen. 1998. Theriogenology. 49(1): 365 Spectra-Physics Products“FCbar ™,” 2 pages, printed Nov. 14, 2002 Spectra-Physics, The SolidState Laser Company “Vanguard 4 Watts of UV from a quasi-cw, all solidstate laser.”, 3 pages, printed Nov. 14, 2002 Spectra-Physics, The SolidState Laser Company, “Vanguard 350-HMD 355, www.spectra-physics.com, 3pp. Spectra-Physics, The Solid State Laser Company, “Vanguard 2000-HM532, www.spectra-physics.com, 3 pp. Time-Bandwidth ® Products,“GE-100-XHP”, www.tbwp.com, 2 pages, Jan. 2002. US National PhaseApplication Number 09/355,461 filed Sep. 17, 1999 Welch G. R., et al.,“Fluidic and optical modifications to a FACS IV for flow sorting of X-and Y- chromosome bearing sperm based on DNA”, Cytometry 17 (suppl. 7):74, 1994

In drafting any claims at any time whether in this application or in anysubsequent application, it should also be understood that the applicanthas intended to capture as full and broad a scope of coverage as legallyavailable. To the extent that insubstantial substitutes are made, to theextent that the applicant did not in fact draft any claim so as toliterally encompass any particular embodiment; and to the extentotherwise applicable, the applicant should not be understood to have inany way intended to or actually relinquished such coverage as theapplicant simply may not have been able to anticipate all eventualities;one skilled in the art, should not be reasonably expected to havedrafted a claim that would have literally encompassed such alternativeembodiments.

Further, if or when used, the use of the transitional phrase“comprising” is used to maintain the “open-end” claims herein, accordingto traditional claim interpretation. Thus, unless the context requiresotherwise, it should be understood that the term “comprise” orvariations such as “comprises” or “comprising”, are intended to implythe inclusion of a stated element or step or group of elements or stepsbut not the exclusion of any other element or step or group of elementsor steps. Such terms should be interpreted in their most expansive formso as to afford the applicant the broadest coverage legally permissible.

1-59. (canceled)
 60. A method of flow cytometry sample processingcomprising the steps of: establishing at least one sheath fluid; flowingsaid at least one sheath fluid into at least two nozzles; injecting atleast one irradiatable sample into said at least one sheath fluid;utilizing at least one shared resource to process said at least oneirradiatable sample; subjecting said irradiatable sample to radiation;exciting said irradiatable sample with said radiation; emittingfluorescence from said excited sample; detecting an amount of saidemitted fluorescence from each particle in said sample; evaluating saidamount of emitted fluorescence from each particle in said sample;selecting an electrical condition to be associated with each particle ofsaid sample in said sheath fluid flow; charging a stream of saidirradiatable sample and sheath fluid based upon deduced properties ofeach particle of said sample in said sheath fluid flow; forming acharged drop; isolating said charged drop from said sheath fluid flow;deflecting said charged drops; sorting said sample; and collecting saidsorted sample. 61-66. (canceled)
 67. A method of flow cytometry sampleprocessing according to claim 60 wherein said step of subjecting saidirradiatable sample to radiation comprises the step of subjecting saidirradiatable sample to a continuous wave laser.
 68. A method of flowcytometry sample processing according to claim 60 wherein said stepsubjecting said irradiatable sample to radiation comprises the steps of:multiply subjecting said irradiatable sample to radiation for a firstamount of time; multiply terminating said radiation of said irradiatablesample for a second amount of time; and multiply exciting saidirradiatable sample with said radiation.
 69. A method of flow cytometrysample processing according to claim 60 wherein said step of utilizingsaid at least one shared resource to process said at least oneirradiatable sample comprises the step of utilizing one radiation sourcefor subjecting said irradiatable sample in said at least two nozzles.70. (canceled)
 71. A method of flow cytometry sample processingaccording to claim 60 and further comprising the step of splitting saidradiation into at least two light beams.
 72. A method of flow cytometrysample processing according to claim 71 wherein said step of splittingsaid radiation into at least two light beams comprises the step ofsubjecting said with a reduced power of radiation than which wasoriginally emitted from a laser source.
 73. A method of flow cytometrysample processing according to claim 72 wherein said step of subjectingsaid with a reduced power of radiation than which was originally emittedfrom a laser source comprises the step of selecting said reduced powerfrom a group consisting of a half, a fourth, and an eighth of saidoriginally emitted power.
 74. A method of flow cytometry sampleprocessing according to claim 60 wherein said step of detecting anamount of said emitted fluorescence from each particle in said samplecomprises the step of quantitatively detecting an amount of said emittedfluorescence from each particle in said sample.
 75. (canceled)
 76. Amethod of flow cytometry sample processing according to claim 74 whereinsaid step of injecting at least one irradiatable sample into said atleast one sheath fluid comprises the step of injecting at least oneirradiatable sperm cells into said at least one sheath fluid and whereinsaid step of quantitatively detecting an amount of said emittedfluorescence from each particle in said sample comprises distinguishingbetween a X chromosome bearing sperm and a Y chromosome bearing spermwherein said X chromosome bearing sperm emits a different fluorescencefrom said Y chromosome.
 77. A method of flow cytometry sample processingaccording to claim 60 wherein said step of sorting said sample comprisesthe step of rapidly sorting said sample.
 78. A method of flow cytometrysample processing according to claim 77 wherein said step of rapidlysorting said sample cells comprises the step of sorting at a rategreater than 500 cells per second.
 79. A method of flow cytometry sampleprocessing according to claim 77 wherein said step of rapidly sortingsaid sample cells comprises the step of sorting at a rate selected froma group consisting of greater than 1000 cells per second; greater than1500 cells per second; greater than 2000 cells per second; and greaterthan 3000 cells per second.
 80. A method of flow cytometry sampleprocessing according to claim 60 and further comprising the step ofutilizing a beam manipulator.
 81. A method of flow cytometry sampleprocessing according to claim 80 wherein said step of utilizing a beammanipulator comprises the step of utilizing a beam manipulator selectedfrom a group consisting of mirrors, deflectors, beam splitters, prisms,refractive objects, lenses and filters. 82-83. (canceled)
 84. A methodof flow cytometry sample processing according to claim 60 wherein saidstep of injecting irradiatable sample comprises the step of stainingsaid sample with fluorescent dye. 85-88. (canceled)
 89. A method of flowcytometry sample processing according to claim 84 wherein said step ofstaining said sample comprises the step of staining said sample for areduced staining time.
 90. A method of flow cytometry sample processingaccording to claim 89 wherein said step of staining for a reduced timecomprises the step of staining said sample for less than about 40minutes.
 91. A method of flow cytometry sample processing according toclaim 89 wherein said reduced staining time is selected from a groupconsisting of less than about 35 minutes; less than about 30 minutes;less than about 25 minutes; less than about 20 minutes; less than about15 minutes; less than about 10 minutes; and less than about 5 minutes.92. A method of flow cytometry sample processing according to claim 60wherein said step of exciting said irradiatable sample with saidradiation comprises the step of sufficiently hitting said sample withsaid radiation to cause said irradiatable sample to emit fluorescence.93-96. (canceled)
 97. A method of flow cytometry sample processingaccording to claim 60 wherein said step of collecting said sorted samplecomprises the step of collecting at least two populations of sampleparticles.
 98. A method of flow cytometry sample processing according toclaim 97 wherein said step of collecting at least two populations ofsample particles comprises the step of collecting a sorted population ofX chromosome bearing sperm and collecting a sorted population of Ychromosome bearing sperm.
 99. A method of flow cytometry sampleprocessing according to claim 97 wherein said step of collecting saidpopulations comprises the step of collecting said populations at a highpurity.
 100. A method of flow cytometry sample processing according toclaim 99 wherein said step of collecting said populations at a highpurity comprises the step of selecting said high purity from a groupconsisting of: greater than 85% purity; greater than 90% purity; greaterthan 95% purity; greater than 96% purity; and greater than 98% purity.101. A method of flow cytometry sample processing according to claim 99wherein said step of collecting said populations at a high puritycomprises the step of a providing a high resolution of said sortedsample.
 102. A method of flow cytometry sample processing according toclaim 101 wherein high resolution of said sorted sample is selected froma group consisting of: greater than 7.0; greater than 7.5; greater than8.0; greater than 8.5; greater than 9.0; and greater than 9.2. 103-104.(canceled)
 105. A method of flow cytometry sample processing accordingto claim 97 wherein said step of collecting at least two populations ofsample particles comprises the step of collecting said populations at ahigh collection rate.
 106. A method of flow cytometry sample processingaccording to claim 105 wherein said high collection rate is selectedfrom a group consisting of: greater than 2400 particles per second;greater than 2600 particles per second; greater than 2900 particles persecond; greater than 3000 particles per second; and greater than 3100particles per second.
 107. A method of flow cytometry sample processingaccording to claim 60 wherein said step of detecting an amount of saidemitted fluorescence from each particle in said sample comprises thestep of detecting at an event rate of between about 10,000 to about60,000 particles per second.
 108. A method of flow cytometry sampleprocessing according to claim 60 wherein said step of subjecting saidirradiatable sample to radiation comprises the step of initiating asensing routine.
 109. A method of flow cytometry sample processingaccording to claim 68 wherein said step of multiply subjecting saidirradiatable sample to radiation for a first amount of time comprisesthe step multiply subjecting said irradiatable sample to radiation for afirst amount of time between about 5 to about 20 picoseconds.
 110. Amethod of flow cytometry sample processing according to claim 68multiply terminating said radiation of said irradiatable sample for asecond amount of time comprises the step of multiply terminating saidradiation of said irradiatable sample for a second amount of timebetween about 0.5 to about 20 nanoseconds.
 111. A method of flowcytometry sample processing according to claim 110 and furthercomprising providing a repetition rate between about 2 to about 10microseconds.
 112. A method of flow cytometry sample processingaccording to claim 111 wherein said step of providing a repetition ratecomprises the step providing a repetition rate between 50-200 MHz. 113.(canceled)
 114. A method of flow cytometry sample processing accordingto claim 84 wherein said step of staining said samples with afluorescent dye comprises the step of minimally staining said sampleswith a fluorescent dye.
 115. A method of flow cytometry sampleprocessing according to claim 114 wherein said step of minimallystaining samples with a fluorescent dye comprises the step of allowingless stain to bind to said sample.
 116. A method of flow cytometrysample processing according to claim 114 wherein said step of minimallystaining said samples with a fluorescent dye comprises the step ofproviding a percentage of stain selected from a group consisting ofabout 90%, about 80%, about 70% and about 60% of a maximum stain. 117.(canceled)
 118. A method of flow cytometry sample processing accordingto claim 60 wherein said step of injecting at least one irradiatablesample into said at least one sheath fluid comprises injecting spermcells selected from a group consisting of mammals, bovine sperm cells,equine sperm cells, porcine sperm cells, ovine sperm cells, camelidsperm cells, ruminant sperm cells, and canine sperm cells.
 119. A methodof flow cytometry sample processing according to claim 60 wherein saidstep of collecting said sorted sample comprises the step of collectingsaid sorted sample in a collector, wherein said collector is selectedfrom the group consisting of multiple containers and a combinedcollector having a individual containers.
 120. A method of flowcytometry sample processing according to claim 119 further comprisingthe step of providing a number of selected containers less than a numberof nozzles.
 121. (canceled)
 122. A method of flow cytometry sampleprocessing according to claim 68 wherein said step of multiplysubjecting said irradiatable sample to radiation for a first amount oftime and said step of multiply terminating said radiation of saidirradiatable sample for a second amount of time comprises the step ofutilizing a pulsed laser.
 123. A method of flow cytometry sampleprocessing according to claim 122 wherein said step of utilizing apulsed laser selected from a group consisting of Nd:YAG and Nd:YVO₄.124. A method of flow cytometry sample processing according to claim 60and further comprising the step of individually controlling said atleast two nozzles.
 125. A method of flow cytometry sample processingaccording to claim 60 and further comprising the step of compositelycontrolling said at least two nozzles.
 126. A mammal produced throughuse of a sorted sperm cells produced with a flow cytometer systemaccording to claim
 118. 127-174. (canceled)
 175. A flow cytometry systemcomprising: at least one sheath fluid port to introduce a sheath fluid;at least one sample injection element having an injection point throughwhich an irradiatable sample may be introduced into said sheath fluid;at least two nozzles located in part below said at least one injectionpoint; an oscillator to which said sheath fluid is responsive; aradiation emitter; a particle sample fluorescence detector; a processingunit connected to said particle sample fluorescence detector; a dropcharge circuit to apply an electrical condition to a stream of saidirradiatable sample cells and sheath fluid; a first and seconddeflection plate each disposed on opposite sides of a free fall area inwhich a drop forms, wherein said first and second deflection places areoppositely charged; and a particle sample collector. 176-231. (canceled)